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Bast fibres are extraxylary sclerenchymatous cells characterized by a noteworthy length and by a cell wall composed of crystalline cellulose. Bast fibres support mechanically the phloem and are used for different industrial applications by the textile and biocomposite sectors. Fibre crops like hemp ( Cannabis sativa ), flax ( Linum usitatissimum ), ramie ( Boehmeria nivea ), jute ( Corchorus olitorius, C. capsularis ), kenaf ( Hibiscus cannabinus ) are therefore important natural resources which can help develop a sustainable economy. Despite the importance of bast fibres, not all the features related to their initiation and growth are fully explored and understood. In this review we will focus on the current knowledge concerning bast fibre initiation and development by using a transcriptomic angle, in the light of the great advances that Next-Generation Sequencing (NGS) has fostered in the last years. We discuss the results obtained recently on different fibre crops and we conclude our survey with a perspective on future molecular studies aimed at valorising neglected fibre crops, e.g. nettle ( Urtica dioica ).

Proper growth of the pollen tube depends on an elaborate mechanism that integrates several molecular and cytological sub-processes and ensures a cell shape adapted to the transport of gametes. This growth mechanism is controlled by several molecules among which cytoplasmic and apoplastic polyamines. Spermine (Spm) has been correlated with various physiological processes in pollen, including structuring of the cell wall and modulation of protein (mainly cytoskeletal) assembly. In this work, the effects of Spm on the growth of pear pollen tubes were analyzed. When exogenous Spm (100 microM) was supplied to germinating pollen, it temporarily blocked tube growth, followed by the induction of apical swelling. This reshaping of the pollen tube was maintained also after growth recovery, leading to a 30-40% increase of tube diameter. Apical swelling was also accompanied by a transient increase in cytosolic calcium concentration and alteration of pH values, which were the likely cause for major reorganization of actin filaments and cytoplasmic organelle movement. Morphological alterations of the apical and subapical region also involved changes in the deposition of pectin, cellulose, and callose in the cell wall. Thus, results point to the involvement of Spm in cell wall construction as well as cytoskeleton organization during pear pollen tube growth.

Various arrays of microtubules are present throughout the plant cell cycle and are involved in distinct functions. Microtubule-associated proteins (MAPs) regulate microtubule dynamics by acting as stabilizers, destabilizers, and promoters of microtubule dynamics. The MAP65 family is a specific group of cross-linkers required for structural maintenance of microtubules. In plants, different isoforms of MAP65 are differentially expressed according to their developmental program. In this work, we analyzed the differential distribution of proteins immunologically related to MAP65-1 during bud development in grapevine (Vitis vinifera L.). First, we annotated the MAP65 genes present in the Vitis genome in order to compare the number and sequence of genes to other species. Subsequently, we focused on a specific isoform (MAP65-1) by characterizing its accumulation and distribution. Proteins were extracted from different organs of Vitis (buds, leaves, flowers, and tendrils), were separated by two-dimensional electrophoresis (2-DE), and were probed by immunoblot with a specific antiserum. We found seven spots immunologically related to MAP65-1, grouped in two distinct clusters, which accumulate differentially according to the developmental stage. In addition, we analyzed the localization of MAP65-1 during three different stages of bud development. Implication of data on the use of different isotypes of MAP65-1 during Vitis development is discussed.

the morphometry of Vitis vinifera seeds from different archaeological sites was studied. preservation status differed between sites. non-invasive and quantitative image analysis based on elliptic Fourier analysis (eFa) established different morphological variations between populations. Molecular analysis was performed to study genetic relationships, using nuclear microsatellite ssrs markers with high polymorphism. Morphometric analysis of archaeological endocarp outlines and allelic profiles of endocarp tegument delineated the general species-specific qualities of the modern cultivar. IANSA 2011 ● II/2 ● 95–100 Claudio Milanesi, francesca Antonucci, Paolo Menesatti, Corrado Costa, Claudia faleri, Mauro Cresti: Morphology and Molecular Analysis of Ancient Grape Seeds 96 2.2 Morphometric analysis the shape analysis of ancient grape seeds for each site, (Firenze, Miranduolo and poggio Bacherina, three samples) was compared with fresh tuscany cultivars from 11 varietals (sangiovese casciano, sangiovese Montalcino, cabernet Bossi, Merlot Bossi, canaiolo Monti in chianti, canaiolo Montalbuccio, trebbiano Montalbuccio, trebbiano tognazza, Malvasia Monti in chianti, albano Montalbuccio and silevstris) and a total of 498 samples. the morphometric method was described in Milanesi et al. 2011 and antonucci et al. 2012. 2.3 DNA analysis ancient seeds were quickly released from the matrix by hcl and hF treatment, which cleaned and removed organic matter (Milanesi et al. 2006), and subsequently exposed overnight to uV-light. specimens were immediately processed for dna extraction under controlled sterile conditions. small pieces of archaeological and modern seeds were divided in two and processed separately for dna extraction and pcr amplification under different sterile laminar-flow hoods. genomic dna was extracted using a method reported by Mulcahy et al. (1993). seven different microsatellite markers (VVMd7, VVMd21, VVMd25, VVMd27, VVMd31, VVMd36, VVs2), generated by Bowers et al. (1999), were amplified. PCR amplification and amplicons detection were performed according to Masi et al. (2001). The amplified loci were cloned using the topo ta clonig kit version F (InVItrogen). plasmids were extracted using the QIAGEN Plasmid Purification kit, and sequenced using the alFexpress autoread sequencing Kit, and analyzed on a polyacrylamide paa gel run and the alFexpressII semi-automated dna sequencer. post run data analysis was performed with the alFwin sequence analyzer 2.00. the generation of the consensus sequence and the pairwise sequence was carried out with dnasys 2.10 software. the analysis was performed in order to observe VVMd7, VVMd21, VVMd25, VVMd27, VVMd31, VVMd36 and VVs2 at least three times.

International audience; An extracellular form of the Ca2+-dependent protein-cross-linking enzyme transglutaminase (TGase) was demonstrated to be involved in the apical growth of Malus domestica pollen tube. Apple pollen transglutaminase and its substrates were co-localized within aggregates on the pollen tube surface, as determined by indirect immuno-fluorescence staining and the in situ cross-linking of fluorescently labeled substrates. Transglutaminase-specific inhibitors and an anti-TGase monoclonal antibody blocked pollen tube growth, whereas incorporation of a recombinant fluorescent mammalian TGase substrate (histidine-tagged green fluorescent protein: His6-Xpr-GFP) into the growing tube wall enhanced tube length and germination, consistent with a role of TGase as modulator of cell wall building and strengthening. The secreted pollen TGase catalyzed the cross-linking of both polyamines (PAs) into proteins (released by the pollen tube) and His6-Xpr-GFP into endogenous or exogenously added substrates. A similar distribution of TGase activity was observed in planta on pollen tubes germinating inside the style, consistent with a possible additional role for TGase in the interaction between the pollen tube and the style during fertilization.

Silver nanoparticles (AgNPs), embedded into a specific exopolysaccharide (EPS), were produced by Klebsiella oxytoca DSM 29614 by adding AgNO3 to the cultures during exponential growth phase. In particular, under aerobic or anaerobic conditions, two types of silver nanoparticles, named AgNPs-EPS(aer) and the AgNPs-EPS(anaer), were produced respectively. The effects on bacterial cells was demonstrated by using Escherichia coli K12 and Kocuria rhizophila ATCC 9341 (ex Micrococcus luteus) as Gram-negative and Gram-positive tester strains, respectively. The best antimicrobial activity was observed for AgNPs-EPS(aer), in terms of minimum inhibitory concentrations and minimum bactericidal concentrations. Observations by transmission electron microscopy showed that the cell morphology of both tester strains changed during the exposition to AgNPs-EPS(aer). In particular, an electron-dense wrapped filament was observed in E. coli cytoplasm after 3 h of AgNPs-EPS(aer) exposition, apparently due to silver accumulation in DNA, and both E. coli and K. rhizophila cells were lysed after 18 h of exposure to AgNPs-EPS(aer). The DNA breakage in E. coli cells was confirmed by the comparison of 3-D fluorescence spectra fingerprints of DNA. Finally the accumulation of silver on DNA of E. coli was confirmed directly by a significant Ag(+) release from DNA, using the scanning electrochemical microscopy and the voltammetric determinations.

Three areas of the 18th century fresco by Sebastiano Conca in the apse of the church (Santissima Annunziata, Siena, Italy) showed signs of deterioration. Twelve small fragments with a total weight of about 15 mg were detached from each of three damaged areas of the fresco and analysed. Element composition of specimens was determined by scanning electron microscopy (SEM) with energy dispersive X-ray analysis (EDX). Some fragments were incubated in mineral medium without organic carbon. Samples from the lower part of the fresco (area A) produced colonies of heterotrophic aerobic bacteria on the carbon available in this medium. Fragments from two other two areas (areas B and C) did not produce any microorganisms. Two strains were isolated from area A and identified as Kocuria erythromyxa strain CV1 and Sphingomonas echinoides strain CV2 by sequencing gene 16S rDNA. When fresco fragments were soaked in mineral medium without carbon source, Sphigomonas echinoides grew rapidly. This strain produced a visible biofilm on the fresco fragments. The mucilage consisted mainly of extracellular polymeric substances (EPS) detected in situ by confocal scanning laser microscopy (CSLM) with Con-A fluorescent lectin and confirmed by transmission electron microscopy (TEM) observations. Ultrastructural analysis of sections of the microorganisms by TEM showed that only strain CV2 adhered to the substrate and caused biological deterioration of the fresco.

A medieval fresco at the Chapel of the Holy nail, Siena was analysed to determine microbiological causes of deterioration of the painted surface or fresco micro-layer. Fragments of fresco a few mm wide were sampled under sterile conditions and some were incubated in mineral medium to isolate microbial species growing on endogenous carbon sources. Quiescent spores of fungi were detected. After 18 h fungal hyphae grew from turgid spores and pH dropped from 5.5 to 5.0, suggesting organic acid production during growth. Light microscopy (LM) and transmission electron microscopy (TEM) were used to study the ultrastructure of the fungi. Molecular studies involving sequencing 18S rDNA led to identification of Penicillium crysogenum. Element concentrations in the painted layer were analysed by scanning electron microscopy (SEM) with energy dispersive X-ray microanalysis of the fresco micro-layer before and after incubation of fragments. The main finding was that total carbon decreased from 26% to 13% while copper increased from 5% to 28%. The high concentration of copper and the acid pH inhibiting bacterial proliferation, indicated P. crysogenum as the main agent of deterioration. Hypotheses are formulated with regard to the high concentrations of copper useful for conservation of fresco substrates.

Polyamines, their analogs, and transglutaminase effects on apical growth of the pollen tube Stefano Del Duca1, Rosa Anna Iorio1, Iris Aloisi1 , Donatella Serafini-Fracassini1, Claudia Faleri 2, Vincenzo Tumiatti3, Anna Minarini3 and Giampiero Cai2 1 Dipartimento di Biologia, Università di Bologna, Via Irnerio 42, Bologna (Italy) 2 Dipartimento Scienze Ambientali, Università di Siena, via Mattioli 4, Siena (Italy) 3 Dipartimento di Scienze Farmaceutiche, Università di Bologna, via Belmeloro 6, Bologna (Italy). e-mail: stefano.delduca@unibo.it Sexual reproduction of flowering plants depends on delivery of the male gamete to the egg cell, which occurs through a polarized growth, located exclusively at the tip of the pollen tube, growing from the pollen grain, distinguished by very fast rates and extended lengths. Pollen germination is characterised by the continuous rebuilding of the cell wall and apical migration of the cytoplasm sustained by cytoskeleton re-organisation. Polyamines (PAs) are essential for cell growth in animals and plants. Early evidence showed that PAs are metabolised and essential factors during pollen tube emergence (Bagni et al. 1981). We investigated the effect of natural and synthetic PAs to check the effect on the apical growth of the pollen tube. We observed that among the natural PAs, spermine inhibited the growth from 10 M onwards. Among the synthetic ones, BD 23, an aromatic derivative of Spm also showed similar effects. The morphology of the tubes showed loss of polarity and enlarged tip. The effects of PAs are related, at least in part to the inhibition of the ROS synthesis, that, at physiological concentration, support the apical growth. As TGase mediates some of the effects of PAs by covalently binding them to proteins, the activity of this enzyme was examined and confirmed by detection of Ca2+-activated TGase activity (Del Duca et al. 1997) and by identification of actin and tubulin as substrates of purified pollen TGase (Del Duca et al. 2009). The enzyme was found in two distinct location: as a soluble and a cell wall associated form (Di Sandro et al. 2010). Fluorescently-labeled of TGase products were essentially present at the apex of pollen tubes, in a region close to the pollen grain and in the pollen grain (Iorio et al. 2008). While the presence and activity of cytoplasmic TGase is likely to be related to the regulation of processes possibly associated with the cytoskeleton activity, the extracellular TGase might provide strength to the pollen tube cell wall during tube migration through the style (Di Sandro et al. 2010). Here, we investigate in detail the localization and distribution of TGase in growing pollen tubes of pear (Pyrus communis). We used specific antibodies that cross-react with TGase of pollen tubes to localize the enzyme in different membrane compartments and in the cell wall of pollen tubes. Use of specific inhibitors indicated that the delivery of extracellular TGase is dependent on both actin filaments and membrane dynamics. Analysis by bidimensional electrophoresis showed that distinct TGase isoforms are associated with different cell compartments. The membrane TGase is likely to be associated with both Golgi-derived structures and the plasma membrane. The distribution of cytoplasmic TGase is dependent on the cytoskeleton while a different TGase form is actively transported via a membrane/cytoskeleton-based transport system and is secreted in the cell wall of pollen tubes, suggesting a Golgi-based exocytosis. Acknowledgements. This research has been supported by the RFO 2010. 1 Bagni, N., Adamo, P. and Serafini-Fracassini, D. (1981) RNA, proteins and polyamines during tube growth in germinating apple pollen. Plant Physiol. 68: 727–730. 2 Folk, J.E., Park, M.H., Chung, S.I., Schrode, J., Lester, E.P. and Cooper, H. (1980) Polyamines as physiological substrates for transglutaminases. J. Biol. Chem.: 255, 3695-3700. 3 Del Duc...

The mechanism by which the sperm activates the egg cell of higher plants is largely unknown. Ca2+—implicated as a second messenger in the response of various plant cells to a wide range of stimuli—is a potential candidate for encoding the information brought by the sperm cell into the angiosperm egg. In higher plant cells the dominant calcium store appears to be the central vacuole; however, in the receptive wheat egg cell few vacuoles can be observed, thus it seems likely that the principal cell organelle performing a pivotal role in regulation of intracellular Ca2+ is the endoplasmic reticulum (ER). To examine this hypothesis, microinjection of the ER-specific fluorescent dicarbocyanine dye, 1,1′-dihexadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate [DiIC16(3)], and low-light level CCD technology were used. Following the injection of an oil drop saturated with DiI, structural changes occurring in the ER during the in planta maturation of the female gamete were visualised. The ER was identified by chlorotetracycline labelling to be the main calcium store in the wheat egg cell. Structural changes occurring in the ER during the in planta maturation of the wheat female gamete led to a polarised ER network in the receptive egg cell. Within 3 min of in vitro sperm-egg fusion, a rapid, transient loss of continuity of the ER occurred, which underlines the significance in fertilisation of the ER structure in the female gamete of wheat.

The smaller isoform of the enzyme glutamic acid decarboxylase (GAD65) is a major islet autoantigen in autoimmune type 1 diabetes mellitus (T1DM). Transgenic plants expressing human GAD65 (hGAD65) are a potential means of direct oral administration of the islet autoantigen in order to induce tolerance and prevent clinical onset of disease. We have previously reported the successful generation of transgenic tobacco and carrot that express immunoreactive, full-length hGAD65. In the present study, we tested the hypothesis that the expression levels of recombinant hGAD65 in transgenic plants can be increased by targeting the enzyme to the plant cell cytosol and by mediating expression through the potato virus X (PVX) vector. By substituting the NH2-terminal region of hGAD65 with a homologous region of rat GAD67, a chimeric GAD671-87/GAD6588-585 molecule was expressed in transgenic tobacco plants. Immunolocalization analysis showed that immunoreactive GAD67/65 was found in the plant cell cytosol. By using a radio-immuno assay with human serum from a GAD65 autoantibody-positive T1DM patient, the highest expression level of the recombinant GAD67/65 protein was estimated to be 0.19% of the total soluble protein, compared to only 0.04% of wild-type hGAD65. Transient expression of wild-type, full-length hGAD65 in N. benthamiana mediated by PVX infection was associated with expression levels of immunoreactive protein as high as 2.2% of total soluble protein. This substantial improvement of the expression of hGAD65 in plants paves the way for immunoprevention studies of oral administration of GAD65-containing transgenic plant material in animal models of spontaneous autoimmune diabetes.

The role of spermine (SM) was studied to verify if SM supplied to Nicotiana tabacum flower can modulate programmed cell death (PCD) of the corolla. SM has strong effects on the development and senescence of excised flowers despite its low physiological levels. The timing and duration of SM treatment is a key factor; SM counteracts PCD (verified by morphological observations, pigment contents and DNA laddering) only in the narrow developmental window of corolla expansion. Before and after, SM promotes PCD. SM exerts its pro-survival role by delaying fresh weight loss, by inhibiting reduction of pigments and finally by preventing DNA degradation. Moreover, SM deeply alters the distribution of the PA-conjugating enzyme transglutaminase (TGase). TGase is present in the epidermis during development, but it sprays also in the cell walls of inner parenchyma at senescence. After SM treatment, parenchyma cells accumulate TGase, increase in size and their cell walls do not undergo stiffening contrarily to control cells. The subcellular localization of TGase has been validated by biolistic-transformation of onion epidermal cells. Results indicated that SM is a critical factor in the senescence of N. tabacum corolla by controlling biochemical and morphological parameters; the lasts are probably interconnected with the action of TGase The role of spermine (SM) was studied to verify if SM supplied to Nicotiana tabacum flower can modulate programmed cell death (PCD) of the corolla. SM has strong effects on the development and senescence of excised flowers despite its low physiological levels. The timing and duration of SM treatment is a key factor; SM counteracts PCD (verified by morphological observations, pigment contents and DNA laddering) only in the narrow developmental window of corolla expansion. Before and after, SM promotes PCD. SM exerts its pro-survival role by delaying fresh weight loss, by inhibiting reduction of pigments and finally by preventing DNA degradation. Moreover, SM deeply alters the distribution of the PA-conjugating enzyme transglutaminase (TGase). TGase is present in the epidermis during development, but it sprays also in the cell walls of inner parenchyma at senescence. After SM treatment, parenchyma cells accumulate TGase, increase in size and their cell walls do not undergo stiffening contrarily to control cells. The subcellular localization of TGase has been validated by biolistic-transformation of onion epidermal cells. Results indicated that SM is a critical factor in the senescence of N. tabacum corolla by controlling biochemical and morphological parameters; the lasts are probably interconnected with the action of TGase.

The Bronze Age archaeological site of Shahr-i Sokhta (30° 39′ N; 61° 24’ E), located today in southeastern Iran, Sistan region, is a special archaeological deposit in which the exceptional preservation of human, plant and animal remains, due to the dry climate of the region, can provide detailed information on one of the first complex proto-urban societies. In recent years, there has been growing interest in changes in local climate and environment as major reasons why the settlement was abandoned about 4000 years ago. Food shortage has been regarded as a direct effect of these changes. No attention has been paid to the potential health hazards associated with ancient urban/domestic pollution, although large garbage deposits have been found in several parts of the site. During excavations in 2007, four soil samples were taken under aseptic conditions at a depth of 1.5–2 m in a stratified deposit sealed by the floor of a small house, dated to the second half of the third millennium BC. Microbiological, palynological, carpological and microanalytical studies were performed on the four soil samples. Site C was identified as the most affected by human activity. Failure of conventional methods of detecting culturable and unculturable microbes in site C indicated the need for specific culture conditions suggested by palynological observations. Since oat seed residues were identified among the archaeobotanical material, oat bran was used as carbon and energy source to make a new medium to revive microbes. Coarsely ground oat bran was sterilized twice and soaked with minimal medium as sole carbon source. About 50 mg of buried soil from site C was added to the medium in cell culture flasks with aerobic and anaerobic stoppers and incubated at 28–30 °C and at 4 °C. After incubation under aerobic and anaerobic conditions, five sporogenic microbes were identified by sequencing 16S rDNA and ITS rRNA regions: a sporogenic strain IRC3 identified as Bacillus sp. was the only isolate under anaerobic conditions, whereas under aerobic conditions four moulds were isolated: Aspergillus flavus IRC1, Penicillium crysogenum IRC2, Cladosporium sp. IRC4, and the psychrotroph Aspergillus restrictus IRC5. Bacillus sp., with 99.7% similarity to Bacillus subtilis , broke down oat bran, producing a gel, while Cladosporium sp., with 99.8% similarity to Cladosporium sphaerospermium, grew on oat bran by synthesizing intracellular lipids. All these microbes are known to spoil food and they are common where there is intense human activity.

In maize, the endosperm region next to the placento-chalaza is characterised by the presence of cells with extensive cell wall ingrowths (transfer cells). Specific genes, collectively named as BETL (for Basal Endosperm Transfer Layer) genes, are expressed in these cells in a way that coincides with the temporal development of the transfer cell layer. The BETL-1 gene encodes a polypeptide of 7 kDa, exclusively expressed in the transfer cell region and possibly involved in the construction of the cell wall. However, the sequence (defensin-like) is also consistent with an antipathogenic role. We found that BETL-1 is strongly associated with the cell wall ingrowths of the endosperm transfer layer. On the other hand, BETL-2 (an antifungal protein) is not bound to the cell wall of transfer cells. The two BETL-2 forms (the 8 kDa propeptide and the final 4.5 kDa peptide) are detected in different subcellular compartments. The propeptide is mostly associated with the membrane fraction of endosperm, while the low molecular weight form is soluble. Vesicles containing the 8-kDa BETL-2 propeptide are located in association with microtubules present in the transfer cell region. Together with previous evidence on the differential distribution of the BETL-2 forms, these data outline the processing pathway of BETL-2, from synthesis in the transfer cells to the site of utilisation in the placento-chalaza region. BETL-4 (which is weakly related to a family of protease inhibitors) is mainly found in the transfer cell layer, and is absent from adjacent regions. Unlike BETL-1, BETL-4 is not a cell wall protein, even though it is associated with the ingrowths of the transfer cell walls. Therefore, BETL proteins show distinct properties of localisation and distribution, which are related to their differential functions in the transfer cells.

Pectin methylesterases (PMEs) were detected in tobacco ( Nicotiana tabacum) pollen tubes grown in vitro. Seven PME isoforms exhibiting a wide isoelectric-point (pI) range (5.3-9.1) were found in crude extracts of pollen tubes. These isoforms were mainly retrieved in supernatants after low- and high-speed separation of the crude extract. Two isoforms, with pIs 5.5 and 7.3 and molecular weight about 158 kDa, were detected by immunoblotting with anti-flax PME antiserum. Localization of pectins and PME isoforms in pollen tubes was investigated by immunogold labelling with JIM5 monoclonal antibodies and anti-flax PME antiserum, respectively. In germinated pollen grains, two PME isoforms were mainly detected in the exine, Golgi apparatus and secretory vesicles. In pollen tubes the same two PME isoforms were distributed along the outer face of the plasma membrane in the vicinity of the inner layer of the cell wall, in the Golgi and around secretory vesicles. In pollen grains, PME isoforms were, in some cases, mixed with acidic pectins in proximity to the outer surface of the plasma membrane. In pollen tubes the presence of PMEs inside secretory vesicles carrying esterified pectins supports the hypothesis that, during pollen tube growth, PMEs could be transferred by secretory vesicles in a precursor form and be activated at the tip where exocytosis takes place.

A series of endosperm transfer layer-specific transcripts has been identified in maize by differential screening of a cDNA library of transcripts at 10 days after pollination. Sequence comparisons revealed among this class of cDNAs a novel, small gene family of highly diverged sequences encoding basal layer antifungal proteins (BAPs). The bap genes mapped to two loci on chromosomes 4 and 10. So far, bap-homologous sequences have been detected only in maize, teosinte and sorghum, and are not present in grasses outside the Andropogoneae tribe. BAP2 is synthesized as a pre-proprotein, and is processed by successive removal of a signal peptide and a 29-residue prodomain. The proprotein can be detected exclusively in microsomal membrane-containing fractions of kernel extracts. Immunolocalization reveals BAP2 to be predominantly located in the placentochalazal cells of the pedicel, adjacent to the basal endosperm transfer layer (BETL) cells, although the BAP2 transcript is found only in the BETL cells. The biological roles of BAP2 propeptide and mature peptide have been investigated by heterologous expression of the proprotein in Escherichia coli, and by tests of its fungistatic activity and that of the fully processed form in vitro. The mature BAP2 peptide exhibits potent broad-range activity against a range of filamentous fungi, including several plant pathogens.

Parenteral and oral administration of autoantigens can induce immune tolerance in autoimmune diseases. Prophylactic therapy based on oral administration of human autoantigens is not, however, feasible when sufficient quantities of candidate autoantigens are not available. Transgenic plants that express high levels of recombinant proteins would allow large quantities of autoantigens to be produced at relatively low costs. In addition, transgenic food would provide a simple and direct method of delivering autoantigens. The production and the characterization of transgenic tobacco and carrot plants expressing human GAD65, a major autoantigen in human insulin-dependent diabetes mellitus (IDDM), is reported. Immunogold labeling and electron microscopy of transgenic tobacco tissue shows the selective targeting of human GAD65 to chloroplast tylacoids and mitochondria. In planta expressed GAD65 has a correct immunoreactivity with IDDM-associated autoantibodies and retains enzymatic activity, a finding that suggests a correct protein folding. In transgenic tobacco and carrot the expression levels of human GAD65 varies between 0.01% and 0.04% of total soluble proteins. Transgenic edible plant organs are now available to study the feasibility of inducing immune tolerance in IDDM animals by oral administration of GAD65.

In this paper we investigated whether a different accuracy in sample cleaning prior to the analysis may affect the elemental composition of unwashed samples of the lichen Xanthoria parietina. To this purpose, samples of this lichen were collected from a remote area of Sardinia (Italy) and randomly divided into two aliquots, one cleaned very carefully (ca. 3-4 h to obtain ca. 200 mg of material for analysis) and the other cleaned with a lower efficiency (ca. 1-2 h). Samples were analysed by ICP-MS for the content of Al, As, Ba, Ca, Cd, Co, Cr, Cu, Fe, K, Mg, Mn, Ni, Pb, V and Zn. The results indicated that an accurate cleaning is effective for reducing the content of Al and Fe, i.e. soil-related elements, but that it has no effect on the concentrations of other elements less represented in the soil.

Nanoplastic debris, resulted from runoff and weathering breakdown of macro- and microplastics, represents an emerging concern for marine ecosystems. The aim of the present study was to investigate disposition and toxicity of polystyrene nanoparticles (NPs) in early development of sea urchin embryos (Paracentrotus lividus). NPs with two different surface charges where chosen, carboxylated (PS-COOH) and amine (PS-NH2) polystyrene, the latter being a less common variant, known to induce cell death in several in vitro cell systems. NPs stability in natural seawater (NSW) was measured while disposition and embryotoxicity were monitored within 48 h of postfertilization (hpf). Modulation of genes involved in cellular stress response (cas8, 14-3-3e, p-38 MAPK, Abcb1, Abcc5) was investigated. PS-COOH forms microaggregates (PDI > 0.4) in NSW, whereas PS-NH2 results are better dispersed (89 ± 2 nm) initially, though they also aggregated partially with time. Their respectively anionic and cationic nature was confirmed by ζ-potential measurements. No embryotoxicity was observed for PS-COOH up to 50 μg mL(-1) whereas PS-NH2 caused severe developmental defects (EC50 3.85 μg mL(-1) 24 hpf and EC50 2.61 μg mL(-1) 48 hpf). PS-COOH accumulated inside embryo's digestive tract while PS-NH2 were more dispersed. Abcb1 gene resulted up-regulated at 48 hpf by PS-COOH whereas PS-NH2 induced cas8 gene at 24 hpf, suggesting an apoptotic pathway. In line with the results obtained with the same PS NPs in several human cell lines, also in sea urchin embryos, differences in surface charges and aggregation in seawater strongly affect their embryotoxicity.

Despite the growing concern over the potential biological impact of nanoparticles (NPs) in the aquatic environment, little is known about their interactions with other pollutants. The bivalve Mytilus sp, largely utilized as a sentinel for marine contamination, has been shown to represent a significant target for different types of NP, including n-TiO2, one of the most widespread in use. In this work, the possible interactive effects of n-TiO2 and 2,3,7,8-TCDD, chosen as models of NP and organic contaminant, respectively, were investigated in Mytilus galloprovincialis. In vitro experiments with n-TiO2 and TCDD, alone and in combination, were carried out in different conditions (concentrations and times of exposure), depending on the target (hemocytes, gill cells and biopsies) and the endpoint measured. Mussels were also exposed in vivo to n-TiO2 (100μgL-1) or to TCDD (0.25μgL-1), alone and in combination, for 96h. A wide range of biomarkers, from molecular to tissue level, were measured: lysosomal membrane stability and phagocytosis in hemocytes, ATP-binding cassette efflux transporters in gills (gene transcription and efflux activity), several biomarkers of genotoxicity in gill and digestive cells (DNA damage, random amplified polymorphic DNA-RAPD changes), lysosomal biomarkers and transcription of selected genes in the digestive gland. The results demonstrate that n-TiO2 and TCDD can exert synergistic or antagonistic effects, depending on experimental condition, cell/tissue and type of measured response. Some of these interactions may result from a significant increase in TCDD accumulation in whole mussel organisms in the presence of n-TiO2, indicating a Trojan horse effect. The results represent the most extensive data obtained so far on the sub-lethal effects of NPs and organic contaminants in aquatic organisms. Moreover, these data extend the knowledge on the molecular and cellular targets of NPs in bivalves. © 2013 Elsevier B.V.

Studies were carried out on structural and cytochemical aspects of the stigma and style of Vitis vinifera . The stigma is of the wet papillate type with a continuous cuticle and pellicle. During the development of the papillae, the cell walls increase in thickness and produce a secretion product constituted of lipids that pass through the wall forming the exudate. The style is solid with a central core of transmitting tissue which has conspicuous intercellular spaces that increase remarkably from the periphery to the centre where the cuticle is present. The intercellular spaces, where the pollen tubes grow, contain a matrix that includes polysaccharides, pectic substances and scattered areas of lipidic nature.

In this study, polyclonal anti-myosin antibodies were used for immunogold labeling of ultrathin sections of pollen tubes ofNicotiana tabacum L. to unravel the ultrastructural localization of myosin associated with the generative cells. Clusters of immunogold particles were consistently found in association with the area of the outer surface of the vegetative cell plasma membrane present around the generative cell. Compared to the generative cell cytoplasm, the nucleoplasm showed higher numbers of gold particles. This is the first direct evidence demonstrating the presence of myosin in the nuclei of the generative cell of flowering plants. The possible implications of these findings are discussed in relation to movement of the generative cell in the pollen tube cytoplasm.

Transglutaminases (TGases) are ubiquitous enzymes that take part in a variety of cellular functions. In the pollen tube, cytoplasmic TGases are likely to be involved in the incorporation of primary amines at selected peptide-bound glutamine residues of cytosolic proteins (including actin and tubulin), while cell wall-associated TGases are believed to regulate pollen tube growth. Using immunological probes, we identified TGases associated with different subcellular compartments (cytosol, membranes, and cell walls). Binding of cytosolic TGase to actin filaments was shown to be Ca2+ dependent. The membrane TGase is likely associated with both Golgi-derived structures and the plasma membrane, suggesting a Golgi-based exocytotic delivery of TGase. Association of TGase with the plasma membrane was also confirmed by immunogold transmission electron microscopy. Immunolocalization of TGase indicated that the enzyme was present in the growing region of pollen tubes and that the enzyme colocalizes with cell wall markers. Bidimensional electrophoresis indicated that different TGase isoforms were present in distinct subcellular compartments, suggesting either different roles or different regulatory mechanisms of enzyme activity. The application of specific inhibitors showed that the distribution of TGase in different subcellular compartments was regulated by both membrane dynamics and cytoskeleton integrity, suggesting that delivery of TGase to the cell wall requires the transport of membranes along cytoskeleton filaments. Taken together, these data indicate that a cytoplasmic TGase interacts with the cytoskeleton, while a different TGase isoform, probably delivered via a membrane/cytoskeleton-based transport system, is secreted in the cell wall of pear (Pyrus communis) pollen tubes, where it might play a role in the regulation of apical growth.

Structural and cytochemical aspects of the pistil of Tibouchina semidecandra Cogn. were studied. The stigma is of the wet-papillate type and is structurally divisible into a papillar zone and a stigmatic zone. The papillar zone consists of loosely arranged papillae which are matchstick-shaped, unicellular, and produce lipid droplets that remain entrapped below the thick cuticle. The bulk of cell volume is made up of large vacuoles rich in tannin. The stigmatic zone consists of layers of secretory cells with dense cytoplasm, actively secreting dictyosomes and numerous rough endoplasmic reticulum (RER) profiles. Free-flowing lipid exudate, produced by these cells, is initially stored in the intercellular spaces, and subsequently extruded out to cover the surface. The style is solid with a core of transmitting tissue traversing its whole length. The transmitting tissue consists of loosely arranged cells with numerous organelles and conspicuous intercellular substance rich in polysaccharides and pectins. Ultrastructural details indicate that the intercellular secretion is accompanied with fraying of the wall component. Both the transverse and longitudinal walls contain plasmodesmata.

The monoclonal antibodies JIM 5 (against unesterified pectin), JIM 7 (against methyl esterified pectin), MAC 207 (against arabinogalactan proteins, AGPs), and JIM 8 (against a subset of AGPs) were utilized singly or in combinations for immunogold labelling of germinated pollen grains and pollen tubes ofNicotiana tabacum. Pectins were localized in the inline of pollen grain, unesterified pectin being more abundant than the esterified one. AGPs were co-localized with pectin in the inline, but were present preferably close to the plasma membrane. In pollen tubes, AGPs, unesterified and esterified pectins were co-localized in the outer and middle layers of the cell wall. The density of the epitopes was not uniform along the length of the pollen tube, but showed alterations. In the pollen tube tip wall esterified pectin was abundantly present, but not AGPs. In the cytoplasm esterified pectin and AGPs were detected in Golgi derived vesicles, indicating their role in the pathway of the cell wall precursors. In the “cell wall” of generative cell only AGPs, but no pectins were localized. The co-localization of pectins and AGPs in the cell wall of pollen grain and pollen tube might play an important role, not only in maintenance of the cell shape, but also in cell-cell interaction during pollen tube growth and development.

A mechanism of mercury detoxification has been suggested by a previous study on Hg bioaccumulation in Manila clams (Ruditapes philippinarum) in the polluted Marano and Grado lagoons and in this study we demonstrate that this event could be partly related to the detoxifying activities of Hg-resistant bacteria (MRB) harbored in clam soft tissues. Therefore, natural clams were collected in six stations during two different periods (winter and spring) from Marano and Grado Lagoons. Siphons, gills and hepatopancreas from acclimatized clams were sterile dissected to isolate MRB. These anatomical parts were glass homogenized or used for whole, and they were lying on a solid medium containing 5 mg l{sup −1} HgCl{sub 2} and incubated at 30 °C. A total of fourteen bacterial strains were isolated and were identified by 16S rDNA sequencing and analysis, revealing that strains were representative of eight bacterial genera, four of which were Gram-positive (Enterococcus, Bacillus, Jeotgalicoccus and Staphylococcus) and other four were Gram-negative (Stenotrophomonas, Vibrio, Raoultella and Enterobacter). Plasmids and merA genes were found and their sequences determined. Fluorescence in situ hybridization (FISH) technique shows the presence of Firmicutes, Actinobacteria and Gammaproteobacteria by using different molecular probes in siphon and gills. Bacterial clumps inside clam fleshmore » were observed and even a Gram-negative endosymbiont was disclosed by transmission electronic microscope inside clam cells. Bacteria harbored in cavities of soft tissue have mercury detoxifying activity. This feature was confirmed by the determination of mercuric reductase in glass-homogenized siphons and gills. -- Highlights: ► We isolated Gram-positive and Gram-negative Hg resistant strains from soft tissues of Ruditapes philippinarum. ► We identify 14 mercury resistant strains by 16S rRNA gene sequences. ► Bacteria in siphon and gill tissues of clams were observed by TEM and identified with different FISH probes. ► Hg-reductase (MerA) activity in glass homogenized clam tissues was also determined.« less

Callose and cellulose are fundamental components of the cell wall of pollen tubes and are probably synthesized by distinct enzymes, callose synthase and cellulose synthase, respectively. We examined the distribution of callose synthase and cellulose synthase in tobacco (Nicotiana tabacum) pollen tubes in relation to the dynamics of actin filaments, microtubules, and the endomembrane system using specific antibodies to highly conserved peptide sequences. The role of the cytoskeleton and membrane flow was investigated using specific inhibitors (latrunculin B, 2,3-butanedione monoxime, taxol, oryzalin, and brefeldin A). Both enzymes are associated with the plasma membrane, but cellulose synthase is present along the entire length of pollen tubes (with a higher concentration at the apex) while callose synthase is located in the apex and in distal regions. In longer pollen tubes, callose synthase accumulates consistently around callose plugs, indicating its involvement in plug synthesis. Actin filaments and endomembrane dynamics are critical for the distribution of callose synthase and cellulose synthase, showing that enzymes are transported through Golgi bodies and/or vesicles moving along actin filaments. Conversely, microtubules appear to be critical in the positioning of callose synthase in distal regions and around callose plugs. In contrast, cellulose synthases are only partially coaligned with cortical microtubules and unrelated to callose plugs. Callose synthase also comigrates with tubulin by Blue Native-polyacrylamide gel electrophoresis. Membrane sucrose synthase, which expectedly provides UDP-glucose to callose synthase and cellulose synthase, binds to actin filaments depending on sucrose concentration; its distribution is dependent on the actin cytoskeleton and the endomembrane system but not on microtubules.

In plant cells, many processes require cooperative action of both microtubules and actin filaments, but proteins mediating interactions between these cytoskeletal members are mostly undiscovered. Here, we attempt to identify such proteins by affinity purification. Cytosol from Nicotiana tabacum (tobacco) pollen tubes was incubated first with actin filaments, and then proteins eluted from the actin were incubated with microtubules, and finally those microtubule-binding proteins were pooled in an active fraction. This fraction bundled actin filaments but not microtubules. However, when the fraction was added to both actin and microtubules, large bundles resulted, containing both polymers, regardless of the order of addition of components. Similar results were obtained when the order of affinity purification was reversed. The four most abundant bands from the fractions were identified from peptide fragments analyzed by mass spectrometry. The same four proteins were identified regardless of the order of affinity purification. The proteins are: homocysteine methyltransferase, phosphofructokinase, pyruvate decarboxylase, and glucan protein synthase (reversibly glycosylated protein). These results suggest the importance of structuring metabolism within the confines of the pollen tube cytoplasm. (C) 2010 Wiley-Liss, Inc

The ultrastructure of Vitis vinifera seeds from different archaeological sites was studied. Preservation status differed between sites. Preliminary investigations of grape seeds from Poggio Bacherina (Chianciano Terme, Siena) and Miranduolo (Chiusdino, Siena) showed collapsed or charred tegument, making this material suitable for morphometric studies only. Rapid-freeze fixation and substitution of grape seeds from Shahr-I Sokhta in Iran and via De’ Castellani in Florence revealed well preserved tegument suitable for chemical and cytochemical analysis. Energy dispersive X-ray microanalysis was used to determine chemical composition. Cytochemical analysis based on fluorescent staining with DAPI suggested the presence of cytoplasm residues.

Sucrose synthase (Sus; EC 2.4.1.13) is a key enzyme of sucrose metabolism in plant cells, providing carbon for respiration and for the synthesis of cell wall polymers and starch. Since Sus is important for plant cell growth, insights into its structure, localization, and features are useful for defining the relationships between nutrients, growth, and cell morphogenesis. We used the pollen tube of tobacco (Nicotiana tabacum) as a cell model to characterize the main features of Sus with regard to cell growth and cell wall synthesis. Apart from its role during sexual reproduction, the pollen tube is a typical tip-growing cell, and the proper construction of its cell wall is essential for correct shaping and direction of growth. The outer cell wall layer of pollen tubes consists of pectins, but the inner layer is composed of cellulose and callose; both polymers require metabolic precursors in the form of UDP-glucose, which is synthesized by Sus. We identified an 88-kD polypeptide in the soluble, plasma membrane and Golgi fraction of pollen tubes. The protein was also found in association with the cell wall. After purification, the protein showed an enzyme activity similar to that of maize (Zea mays) Sus. Distribution of Sus was affected by brefeldin A and depended on the nutrition status of the pollen tube, because an absence of metabolic sugars in the growth medium caused Sus to distribute differently during tube elongation. Analysis by bidimensional electrophoresis indicated that Sus exists as two isoforms, one of which is phosphorylated and more abundant in the cytoplasm and cell wall and the other of which is not phosphorylated and is specific to the plasma membrane. Results indicate that the protein has a role in the construction of the extracellular matrix and thus in the morphogenesis of pollen tubes.

The pollen tube exhibits cytoplasmic streaming of organelles, which is dependent on the actin-myosin system. Although microtubule-based motors have also been identified in the pollen tube, many uncertainties exist regarding their role in organelle transport. As part of our attempt to understand the role of microtubule-based movement in the pollen tube of tobacco, we investigated the cooperation between microtubules and actin filaments in the transport of mitochondria and Golgi vesicles, which are distributed differently in the growing pollen tube. The analysis was performed using in vitro motility assays in which organelles move along both microtubules and actin filaments. The results indicated that the movement of mitochondria and Golgi vesicles is slow and continuous along microtubules but fast and irregular along actin filaments. In addition, the presence of microtubules in the motility assays forces organelles to use lower velocities. Actin- and tubulin-binding tests, immunoblotting and immunogold labeling indicated that different organelles bind to identical myosins but associate with specific kinesins. We found that a 90 kDa kinesin (previously known as 90 kDa ATP-MAP) is associated with mitochondria but not with Golgi vesicles, whereas a 170 kDa myosin is distributed on mitochondria and other organelle classes. In vitro and in vivo motility assays indicate that microtubules and kinesins decrease the speed of mitochondria, thus contributing to their positioning in the pollen tube.

The method we have developed allows the cytoplasm of a single fossil cell to be sufficiently preserved as to be distinguished by fluorescence microscopy. Analysis of fossil DNA involved PCR amplification of the spacer re- gion between rrn5 and trnR genes in the chloroplast genome. The DNA and consensus sequences, obtained from the same PCR amplification fragment, were analysed by BLAST search and CLUSTAL W multiple alignment software.

The pollen surface consists of an outermost coat and an underlying wall. It makes the initial contact with the stigma surface during sexual reproduction. To date, only two proteins have been identified from the maize pollen coat. Zea m 1 (beta-expansin 1) is the major group-1 allergen in maize pollen, but its presence and localization in the pollen coat and wall has not yet been explored. In the present study, immunoblot analysis using an antibody directed against group-1 allergen revealed that a small amount of Zea m 1 exists in the pollen coat fraction prepared using a diethyl ether wash. Immunogold labeling also showed that the extracellular localization of Zea m 1 was mainly restricted to the tectum and the foot layer of the exine (the outer pollen wall), and gold particles immunolabelling Zea m 1 were unevenly dispersed throughout the pollen coat and wall. Moreover, a substantial amount of Zea m 1 was localized in the cytoplasm of the pollen interior. The presence of Zea m 1 in the pollen coat and wall suggests that Zea m 1 may play a potential role in pollen germination on the stigma.

We successfully combined ultrastructure and chloroplast DNA analysis for single specimens of Concentricystis isolated from Holocene sediment by a palynology method based on filtration, progressive sieving and percoll-gradient sedimentation to concentrate fossil cells. We compared our method with the traditional harsh chemical treatment commonly used to isolate fossil pollen and spores. With our method, the cytoplasm of Concentricystis was sufficiently preserved to distinguish several cell structures by light and electron microscopy. DNA analysis of Concentricystis involved PCR amplification using specific primers for a spacer region of the chloroplast genome. Significant homologies were found with the Angiosperm chloroplast genome by BLAST DNA search for PCR product sequences.

Puroindolines, for years largely investigated for their involvement in wheat kernel hardness, have recently attracted attention thanks to their possible role as antimicrobial proteins. With the aim to enhance our knowledge of these proteins we studied their localization in the kernel, and their antimicrobial activity in vitro against six different bacterial strains. Immunolocalization showed that both the PINs are strongly concentrated in the aleurone layer, but also highly present in the endosperm. Interestingly we observed that puroindolines not only have the same spatial distribution in the kernel, they are also always found co-localized. Their co-localization suggests that they could cooperate in defending the plant against pathogens. We therefore tested antimicrobial activity of PINA and PINB, and a putative synergism between these proteins. The results showed that the two polypeptides can in vitro inhibit growth of all the bacteria tested; furthermore when combined together they are able to enhance each other's toxicity. In view of their antimicrobial activity and of their natural presence in Triticum aestivum wheat flour, puroindolines look promising antibacterial agents and thus deserve further studies aimed at establishing their possible future applications in fields of food and health care. Since PINs were still detectable in bakery products, these proteins may be promising tools in investigating natural ways of food preservation.

Sk2 protein is the most abundant member of the pistil-specific proteins of Solanum tuberosum. Sk2 protein has been localized by use of a polyclonal antibody (anti-Sk2) in the pistils of four clones of Solanum tuberosum. In the stigmas Sk2 protein accumulates to a high level in the cytoplasm of the internal secretory cells underlying the papillae one day prior to anthesis. In styles, the intercellular matrix of the transmitting tissue cells is intensely labelled by anti-Sk2. Sk2 protein is present in all four clones and shows the same labelling pattern. The possible role of the Sk2 protein in pollen tube growth is discussed.

Self-incompatibility has been investigated in the cultivar ‘Abbé Fétel’ (Pyrus communis) in order to assign a role to transglutaminase (TGase), an enzyme able to post-translationally forming cross-links among proteins. This enzyme is localised in the pollen cytoplasm where it regulates the polymerisation of cytoskeleton proteins. An extracellular form is also necessary for pollen tube growth. In the self-pollinated style of Abbè Fétel (A x A, incompatible system), the activity of TGase increased when the pollen tube stopped its growth inside the style and the enzyme was immuno-localised around the tube tip. On the contrary in Abbé Fétel styles pollinated with Williams pollen (A x W, compatible system), TGase activity decreased during pollen germination. The TGase gene has been cloned and sequenced from Abbé Fétel pollen and style showing that it shares a high homology with a sequence of apple, whose genome has been recently published, which presents the typical TGase catalytic sequence, and with the TGase of Arabidopsis. This enzyme was expressed during pollen germination inside the style to a similar extent in both systems (A x A and A x W) showing that it is only the activity to have been stimulated by some factor dependent on SI. A molecular approach to clone one of the S-RNase alleles of the cv Abbé Fétel has been performed. This protein, supplied to pollen in the germination medium, was able to inhibit the growth of 50% of the tubes of the homologous pollen. This approach will allow us to verify if an interaction between the two enzymes will occur during SI, as TGase, at least in animal cells, is also able to act as disulphide isomerase modifying the RNase structure.