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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. [ABSTRACT FROM AUTHOR]

The male germ cells of angiosperm plants are neither free-living nor flagellated and therefore are dependent on the unique structure of the pollen grain for fertilization. During angiosperm male gametogenesis, an asymmetric mitotic division produces the generative cell, which is completely enclosed within the cytoplasm of the larger pollen grain vegetative cell. Mitotic division of the generative cell generates two sperm cells that remain connected by a common extracellular matrix with potential intercellular connections. In addition, one sperm cell has a cytoplasmic projection in contact with the vegetative cell nucleus. The shared extracellular matrix of the two sperm cells and the physical association of one sperm cell to the vegetative cell nucleus forms a linkage of all the genetic material in the pollen grain, termed the male germ unit. Found in species representing both the monocot and eudicot lineages, the cytoplasmic projection is formed by vesicle formation and microtubule elongation shortly after the formation of the generative cell and tethers the male germ unit until just prior to fertilization. The cytoplasmic projection plays a structural role in linking the male germ unit, but potentially plays other important roles. Recently, it has been speculated that the cytoplasmic projection and the male germ unit may facilitate communication between the somatic vegetative cell nucleus and the germinal sperm cells, via RNA and/or protein transport. This review focuses on the nature of the sperm cell cytoplasmic projection and the potential communicative function of the male germ unit. [ABSTRACT FROM PUBLISHER]

The growth of pollen tubes is supported by the continuous supply of secretory vesicles in the tip area. Movement and accumulation of vesicles is driven by the dynamic interplay between the actin cytoskeleton and motor proteins of the myosin family. A combination of the two protein systems is also responsible for the bidirectional movement of larger organelle classes. In contrast, the role of microtubules and microtubule-based motors is less clear and often ambiguous. Nevertheless, there is evidence which shows that the pollen tube contains a number of microtubule-based motors of the kinesin family. These motor proteins are likely to be associated with pollen tube organelles and, consequently, they have been hypothesized to participate in the distribution of organelles during pollen tube growth. Whether microtubule motor proteins take part in either the transport or positioning of organelles is not known for sure, but there is evidence for this second possibility. This review will discuss the current knowledge of microtubule-based motor proteins (including kinesins and hypothetical dyneins) and will make some hypothesis about their role in the pollen tube. [ABSTRACT FROM AUTHOR]

Organelle movement is an evident feature of pollen tubes and is essential for the process of tube growth because it enables the proper distribution of organelles and the accumulation of secretory vesicles in the tube apex. Organelles move along the actin filaments through dynamic interactions with myosin but other proteins are probably responsible for control of this activity. The role of microtubules and microtubule-based motors is less clear and somewhat enigmatic. Nevertheless, the pollen tube is an excellent cell model in which to study and analyse the molecular mechanisms that drive and control organelle motility in relation to plant cell expansion. Current knowledge and the main scientific discoveries in this field of research over the last 20 years are summarized here. Future prospects in the study of the molecular mechanisms that mediate organelle transport and vesicle accumulation during pollen tube elongation are also discussed. [ABSTRACT FROM PUBLISHER]

Using rapid fixation and freeze substitution method, the ultrastructure of the cytoplasm, including the membrane system, of Betula pendula pollen is well preserved. The cytoplasm of Betula pollen appears compact and organelles such as Golgi and RER are not yet demonstrating any secretory activities. The cytoplasm streaming is not established because the pollen grains are not completely hydrated and the microfilaments are still polymerized in bundless. The cytoplasm of the generative cell is well preserved including the cell wall like structure and cytoskeletal complexes present in the cell. Moreover the application of rapid freeze fixation of microspores and pollen grains of Betula showed clear details of ultrastructure of the Zwischenkörper (Z) at the aperture sites and the Z-layer (ZL) between the apertures. They are mainly composed of dense helical units that extend from the plasmalemma. Rapid freezing improves to a great extent the fixation of the delicate cell structure of the tapetum. After freeze fixation and substitution it is possible to observe radial helical units similar to those of the Zwischenkörper, throughout the thick coat of tapetal cells. [ABSTRACT FROM AUTHOR]

Presents a study on structure and development of flagellated and nonflagellated sperm in plants. Methods and materials employed; Results of the study.

The article presents an obituary for Hans Ferdinand Linskens, an assistant professor at the University of Cologne.

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. [ABSTRACT FROM AUTHOR]

The pollen tube is fundamental for the reproduction of seed plants. Characteristically, it grows relatively quickly and uni-directionally ('polarized growth') to extend the male gametophyte to reach the female gametophyte. The pollen tube forms a channel through which the sperm cells move so that they can reach their targets in the ovule. To grow quickly and directionally, the pollen tube requires an intense movement of organelles and vesicles that allows the cell's contents to be distributed to sustain the growth rate. While the various organelles distribute more or less uniformly within the pollen tube, Golgi-released secretory vesicles accumulate massively at the pollen tube apex, that is, the growing region. This intense movement of organelles and vesicles is dependent on the dynamics of the cytoskeleton, which reorganizes differentially in response to external signals and coordinates membrane trafficking with the growth rate of pollen tubes. Giampiero Cai [ABSTRACT FROM AUTHOR]

The cell wall of pollen tubes is organized in both spatial and temporal order to allow the pollen tube to grow according to external conditions. The deposition of methyl-esterified and acid pectins in addition to callose/cellulose occurs according to a series of temporally succeeding events. In this work, we attempted to determine how the composition of the external growth medium (in terms of osmolarity) could affect the deposition of cell wall components. Pollen tubes of tobacco were grown in a hypotonic medium and then analyzed for the distribution of pectins and callose/cellulose [as well as for the distribution of the enzyme callose synthase (CALS)]. The data indicate that pollen tubes grown in a hypotonic medium show changes of the initial growth rate followed by modification of the deposition of acid pectins and, to a lesser extent, of CALS. These observations indicate that, under the osmolarity determined by the growth medium, pollen tubes adapt their cell wall to the changing conditions of growth. [ABSTRACT FROM AUTHOR]

Barley (Hordeum vulgare L.) is one of the main domesticated cereals. For this reason, barley seeds have been found in numerous archaeological sites, and since the mid-19th century have been available in numerous natural museum collections. About a hundred years ago samples were collected in the African countries of Eritrea and Cyrenaica (now Libya), and have been preserved as ex-situ in the museum collection "L'Orientale" of the University of Naples. The varieties of contemporary barley selected for comparative analysis were grown in Tuscany and are used in the production of craft beer. To ascertain their vitality, the ancient and contemporary seeds were placed in Petri dishes to hydrate under a sterile hood at room temperature after a sterilization procedure. Morphological and ultrastructural observations performed on the aleurone cells of the ancient samples presented vital cells. The extraction and purification of DNA from seeds produced results while the genotype comparison of ancient and contemporary barley varieties enabled the construction of a dendrogram of similarity, useful in describing barley from museum genetic heritage collections and in providing a molecular imprint of extant varieties. [ABSTRACT FROM AUTHOR]

Describes an experiment, which demonstrated that pollen tube organelles use kinesin-related motor proteins to move along microtubules. Background on organelle movement; Role of the interactions between actin filaments and membranes in plant-cell movement; Analysis of the relationships between microtubules and organelle transport in the pollen tube; Significance of cytosolic proteins in promoting the transport of pollen tube organelles along microtubules.

In the late Roman Republic period (2nd–1st century BC), in the area of San Giovanni on Elba Island, previously subject to intense extraction of iron ore, a rustic villa was established by Marco Valerio Messalla, a supreme Roman magistrate. The foundations of the walls were discovered and excavated by an archaeological mission. Palaeobotanical analysis of a set of stratigraphic layers was performed. Palynological slides showed remains of palynomorphic and non-pollen objects, while data combined with anthracological investigations confirmed the hypothesis that in the 1st century AD the villa was destroyed by a fire that created a compact crust under which were discovered four broken Roman amphorae containing about five hundred apple seeds. Comparisons of archaeological and fresh seeds from reference collections showed discontinuous morphology except for one group of archaeological samples. DNA was isolated from seeds that had well-preserved embryos in all groups. DNA extracts from archaeological, wild and modern domestic seeds (controls) were amplified by PCR and tested with SSR molecular markers, followed by genome analysis. [ABSTRACT FROM AUTHOR]

The pollen coat, which covers the exine wall of pollen, is essential for initial sexual contact and thus successful fertilization in flowering plants. Pollen coat proteins (PCPs) not only mediate species specificity but are also needed for pollen-stigma recognition and pollen germination on the stigma. Maize ( Zea mays L.) is one of the most common cereal crops in the world. To date, only a few PCPs from maize have been identified and characterized. In the present study, we extracted the pollen coat fraction from maize inbred line B73 with chloroform and purified the PCPs via a phenol-based protocol prior to two-dimensional gel electrophoresis (2-DE). By proteomic analysis, 26 protein spots were successfully identified and classified into 12 unique proteins. The protein composition of the maize pollen coat is distinctly different from those of the dicot plants Arabidopsis and rapeseed. Of the proteins identified in this study, eight (including profilins, caleosin, Zea m 2, β-expansin-10, exopolygalacturonase, Rho GDP-dissociation inhibitor 1, Ras-related protein Rab-2-A, and putative subtilase) had not previously been observed in the maize pollen coat. Bioinformatic analysis showed that nine of the PCPs were secreted proteins and that most of them were extracellular. These PCPs are potentially involved in pollen germination and tube growth. The current study extracted and identified the pollen coat proteins of maize, one of the most important crops throughout the world. Proteome profiling of the maize pollen coat revealed many novel protein components potentially involved in pollen-stigma interactions and pollen germination. Our results provide basic knowledge and further the functional characterization of PCPs in wind-pollinated species such as maize. [ABSTRACT FROM AUTHOR]

Potatoes ( Solanum tuberosum L.) from the high Tiber valley area (TVA; Tuscany, Italy), have been sampled and analyzed for selected mineral content (Na, Mg, K, Ca, and Mn, Fe, Cu and Zn) and vitamin C, the year 2012; some samples from 2011 and 2013 crops were also collected and analyzed. The varieties were Daytona (DAY), Kennebec (KEN), Sifra (SIF) and Volare (VOL). Control samples consisted of mixed commercial varieties from the local market, namely C1, C2 and C3. The low content of sodium, especially for KEN (46 ± 3 mg/kg FD (freeze dry), year 2012) and SIF (47 ± 3) (VOL (55 ± 3) and DAY (61 ± 3) have a little higher values) is worth of note and in agreement with the scarce concentration of Na in the soil (291 ± 12 mg/kg DM). Magnesium was abundant in KEN (1434 ± 75 mg/kg FD, year 2012) and VOL (1334 ± 70). The content of K for DAY and KEN (13,147 ± 900 and 13,185 ± 900 mg/kg FD) was higher than for VOL and SIF; whereas Ca was in the range 340 ± 16–490 ± 28 mg/kg FD. The contents of Cu and Zn were higher in KEN (8.1 ± 0.3 and 25 ± 1 mg/kg FD) when compared to the other varieties and controls. The content of vitamin C is high for KEN and SIF and decreased significantly upon cooking (50% for KEN). [ABSTRACT FROM AUTHOR]

During in vitro fertilization of wheat (Triticum aestivum, L.) in egg cells isolated at various developmental stages, changes in cytosolic free calcium ([Ca2+]cyt) were observed. The dynamics of [Ca2+]cyt elevation varied, reflecting the difference in the developmental stage of the eggs used. [Ca2+]cyt oscillation was exclusively observed in fertile, mature egg cells fused with the sperm cell. To determine how [Ca2+]cyt oscillation in mature egg cells is generated, egg cells were incubated in thapsigargin, which proved to be a specific inhibitor of the endoplasmic reticulum (ER) Ca2+-ATPase in wheat egg cells. In unfertilized egg cells, the addition of thapsigargin caused an abrupt transient increase in [Ca2+]cyt in the absence of extracellular Ca2+, suggesting that an influx pathway for Ca2+ is activated by thapsigargin. The [Ca2+]cyt oscillation seemed to require the filling of an intracellular calcium store for the onset of which, calcium influx through the plasma membrane appeared essential. This was demonstrated by omitting extracellular calcium from (or adding GdCl3 to) the fusion medium, which prevented [Ca2+]cyt oscillation in mature egg cells fused with the sperm. Combined, these data permit the hypothesis that the first sperm-induced transient increase in [Ca2+]cyt depletes an intracellular Ca2+ store, triggering an increase in plasma membrane Ca2+ permeability, and this enhanced Ca2+ influx results in [Ca2+]cyt oscillation. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: Truffles, such as Tuber magnatum and Tuber melanosporum, are greatly appreciated throughout the world, both as fresh fruiting bodies and as ingredients in processed products. Diagnostic methods are therefore required to check the identity of truffles in this kind of food. The present paper reports the application of microscopic and molecular techniques to authenticate truffle species in commercial products. Through the application of microscopic techniques, it has been possible to distinguish spores in a cream that could be ascribed to the truffle species (T. magnatum, the highest priced truffle) declared on the label and also spores of a less prized and aromatic truffle that was not indicated on the label. Good quality DNA was quickly obtained in a few hours using a kit generally employed for DNA extraction from soil. A new primer pair was developed to authenticate T. magnatum in commercial products and it was employed in a quantitative PCR assay (qPCR). T. melanosporum, which was neither indicated on the label nor recognized in the processed products containing truffles, was also detected in a cream and its amount was quantified by qPCR. This method can therefore be used to detect fraudulent practices and to protect the consumer of truffle delicacies. [Copyright &y& Elsevier]

Background: Cinta Senese (CS) is an autochthonous Tuscan breed, which risked extinction since the '60s. Results: Monitoring the genetic variability of the actual population by use of DNA molecular markers is essential to address a correct breeding policy, finalized to obtain the race preservation and its fitness in the future. 17 SSRs autosomal markers and 1 associated to the X chromosome were used to genotype 86 individuals belonging to the CS and 12 belonging to two main white races Landrace (L), Large White (LW) and crosses between LW and L and L and CS widespread in Tuscany and used in the recent past to obtain hybrids with the CS. Conclusions: A dendrogram of similarity measures the relative genetic distance between individuals in the population. Data show that CS pigs have a distinct genotype from L, LW, LW x L and L x CS. [ABSTRACT FROM AUTHOR]

Abstract: Morphological analysis by light and electron microscopy was conducted on wine residues from second century Roman amphorae found in Myrmekion (Ukraine). The results of the archaeological samples were compared with residues formed in bottles of wine from Tuscan vineyards, corked between 1969 and 1977 without filtration and enzyme or biochemical processing. Staining and histochemical observation of the archaeological and recent residues detected nucleic acids. Molecular analysis was also performed using nuclear microsatellite SSRs markers having high polymorphism to study genetic relationships. Genotype profiles of archaeological and recent residues were compared with contemporary cultivars in a data bank. Low homology of genotype profiles of all residues, and oral evidence, confirmed the presence of autochthonous varieties in recent wines and enabled indirect assessment of varieties detected in the archaeological material. The results confirmed that the archaeological material could be related to Sangiovese and indicated Roman wine trade as far afield as Ukraine, whereas the recent residues provided evidence of disappearing native Tuscany cultivars like Gorgottesco, Mammolo, Verdello, Rossone and Tenerone, used for table wines until the late seventies. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

Abstract: Preliminary studies of historical sources and remote sensing were used to identify ancient olive trees near archaeological sites and heritage buildings in the Orcia Valley (Siena, Italy). Distinctive characters were assessed by traditional pomological observation. Trees with similar characters were selected on the basis of the features of endocarps, the only structure that survives aerobic deterioration and conserves useful botanical information for centuries. Non-invasive morphometric analysis of endocarp size and shape established morphological variations in individuals of different populations. Plastid organization in the endocarp and location of DNA in the endocarp tegument were detected by morphological and ultrastructural observations using light and electron microscopy. Cytoplasmic markers with high polymorphism were used to test similarity of endocarp and leaf DNA within individuals and to confirm low variability and minimal divergence between individuals. The ancient trees studied showed the same allelic profiles and therefore belonged to a distinct cultivar. The traditional pomological descriptions of the trees, leaves and fruits, morphometric analysis of size, and shape elliptic Fourier analysis of endocarp outline, ultrastructural observations and allelic profiles of endocarp tegument delineated the general species-specific qualities of the cultivar “olivastra Seggianese” of the Orcia Valley. [ABSTRACT FROM AUTHOR]

collection of 1005 grapevine accessions was genotyped at 34 microsatellite loci (SSR) with the aim of analysing genetic diversity and exploring parentages. The comparison of molecular profiles revealed 200 groups of synonymy. The removal of perfect synonyms reduced the database to 745 unique genotypes, on which population genetic parameters were calculated. The analysis of kinship uncovered 74 complete pedigrees, with both parents identified. Many of these parentages were not previously known and are of considerable historical interest, e.g. Chenin blanc (Sauvignon × Traminer rot), Covè (Harslevelu selfed), Incrocio Manzoni 2-14 and 2-15 (Cabernet franc × Prosecco), Lagrein (Schiava gentile × Teroldego), Malvasia nera of Bolzano (Perera × Schiava gentile), Manzoni moscato (Raboso veronese × Moscato d'Amburgo), Moscato violetto (Moscato bianco × Duraguzza), Muscat of Alexandria (Muscat blanc à petit grain × Axina de tres bias) and others. Statistical robustness of unexpected pedigrees was reinforced with the analysis of an additional 7-30 SSRs. Grouping the accessions by profile resulted in a weak correlation with their geographical origin and/or current area of cultivation, revealing a large admixture of local varieties with those most widely cultivated, as a result of ancient commerce and population flow. The SSRs with tri- to penta-nucleotide repeats adopted for the present study showed a great capacity for discriminating amongst accessions, with probabilities of identity by chance as low as 1.45 × 10 and 9.35 × 10 for unrelated and full sib individuals, respectively. A database of allele frequencies and SSR profiles of 32 reference cultivars are provided. [ABSTRACT FROM AUTHOR]

Abstract: Painted surfaces in the Chapel of the Holy Nail in the medieval ex-hospital of Siena show five areas of oxidation and blackening. Most were treated with Paraloid B72 acrylic resin 40 years ago. To study deterioration of the painted surfaces by scanning microscopy with X-ray dispersion microanalysis, fragments obtained from the five areas were analyzed directly and after hydration. Hydrated fragments treated with Paraloid showed a compact, shiny, impermeable surface. The acrylic resin forms a layer that inhibits interaction of the painting with the environment, including transpiration and gaseous exchange. Element composition of paint layers of different colours revealed gold, iron (oxides in ochre) and lead (oxide) tempered with lime and/or gypsum. A few microbial cells were found on all fragments. To study biological deterioration in the five areas, fragments were hydrated and incubated in minimal culture medium without added carbon source to select bacteria capable of using carbon sources in the medieval fresco. Analysis did not reveal any spores or hyphae, and excluded physical and mechanical damage. Bacteria of the genus Bacillus were isolated only from untreated samples. Paraloid resin forms an inert film over the mural, preventing access to microbes able to use nutrient sources in the painted layer. The screening of 16S rRNA libraries from enrichment cultures showed wide phylogenetic diversity. Forty-four percent of the clones retrieved from the clone library were affiliated with the order Firmicutes, confirming the prevalence of aerobic spore-formers among the colonizing microflora. Firmicutes therefore presumably produced extracellular material which made water available to other bacteria which may have converted thiosulphate in the medieval tempera to sulphur globules. Sulphur is a known oxidant of metallic pigments, in this case aluminium, which may explain the blackening of the untreated sample. [Copyright &y& Elsevier]

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 hi the construction of the extracellular matrix and thus in the morphogenesis of pollen tubes. [ABSTRACT FROM AUTHOR]

Summary: 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 (β-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. [Copyright &y& Elsevier]

Abstract: 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 15mg 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. [Copyright &y& Elsevier]

Exhibiting rapid polarized growth, the pollen tube delivers the male gametes into the ovule for fertilization in higher plants. To get an overall picture of gene expression during pollen germination and pollen tube growth, we profiled the transcription patterns of 1,536 pollen cDNAs from lily (Lilium longiflorum) by microarray. Among those that exhibited significant differential expression, a cDNA named lily ankyrin repeat-containing protein (LlANK) was thoroughly studied. The full-length LlANK cDNA sequence predicts a protein containing five tandem ankyrin repeats and a RING zinc-finger domain. The LlANK protein possesses ubiquitin ligase activity in vitro. RNA blots demonstrated that LlANK transcript is present in mature pollen and its level, interestingly contrary to most pollen mRNAs, up-regulated significantly during pollen germination and pollen tube growth. When fused with green fluorescent protein and transiently expressed in pollen, LlANK was found dominantly associated with membrane-enclosed organelles as well as the generative cell. Overexpression of LlANK, however, led to abnormal growth of the pollen tube. On the other hand, transient silencing of LlANK impaired pollen germination and tube growth. Taken together, these results showed that LlANK is a ubiquitin ligase associated with membrane-enclosed organelles and required for polarized pollen tube growth. [ABSTRACT FROM AUTHOR]

Abstract: 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 18h 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. [Copyright &y& Elsevier]

The organization and function of microtubules in plant cells are important in many developmental stages. Connections between microtubules and the endomembrane system of plant cells have been discovered by microscopy, but the molecular characteristics of these relationships are mostly unknown except for a few cases. Using two antibodies raised against microtubule-associated proteins (MAPs) from maize, we have identified two polypeptides that share properties of the MAP family in the pollen tube of Nicotiana tabacum. The two polypeptides (with an apparent Mr of 161 and 90 kDa) bind efficiently to animal and plant microtubules and are found in association with the cellular membranes of the pollen tube, from which they can be solubilized with a zwitterionic detergent. One of these proteins has been purified and shown to promote the assembly of tubulin and, to a lesser extent, the bundling of microtubules. Subcellular fractionation indicated that the two proteins are associated with the plasma membrane compartment. The two proteins are found to co-localize in situ with cortical microtubules in the vegetative cytoplasm of tobacco pollen tubes; co-localization is also evident in the generative cell. According to these data, both the 161 and 90 kDa polypeptides are likely to mediate the interactions between the plasma membrane and microtubules in pollen tubes. In addition, functional data indicate that these MAP-like proteins take part in the process of microtubule assembly and reorganization occurring during cell growth. The evidence that both proteins associate with different cellular compartments also suggests a broad-spectrum role in mediating the dynamic relationships between microtubules and plant cell membranes. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

gaMS-2 is a gametophytic male-sterile mutant of maize, with sterile pollen grains developmentally blocked at the binucleate stage. To characterise differentially expressed proteins in gaMS-2 pollen, we compared protein profiles of anthers and mature pollen from heterozygous GaMS-2/gaMS-2 plants and wild type (wt) plants by two-dimensional electrophoresis (2-DE). A basic protein present at a greatly reduced level in GaMS-2/gaMS-2 anthers was subsequently identified by tandem mass spectrometry as Zea m 1 (a glycoprotein of 31 kDa), the major group-1 allergen of maize pollen and a member of the β-expansin 1 family. Moreover, we show that Zea m 1 can be deglycosylated by peptide N-glycosidase F. After deglycosylation, four major isoforms—Zea m 1a (more acetic), Zea m 1b, Zea m1c and Zea m 1d (more basic)—can be discriminated in wt anther in 2-DE immunoblots probed with a monoclonal antibody against the group-1 pollen allergen, whereas all the isoforms, especially Zea m 1a, exist at reduced levels in GaMS-2/gaMS-2 anthers. Furthermore, the reduced Zea m 1 accumulation in the mutant appears to occur in immature pollen but not in anther sporophytic tissues. Finally, we separated sterile pollen grains (at the mononucleate stage) from fertile ones using 42% Percoll solution, and found that Zea m 1 is barely detectable in sterile pollen grains. Together, our results indicate that a reduced Zea m 1 level is associated with the sterile phenotype of gaMS-2. [ABSTRACT FROM AUTHOR]

In an attempt to dissect endocytosis in Nicotiana tabacum L. pollen tubes, two different probes positively or negatively charged nanogold were employed. The destiny of internalized plasma membrane domains, carrying negatively or positively charged residues, was followed at the ultrastructural level and revealed distinct endocytic pathways. Time-course experiments and electron microscopy showed internalization of subapical plasma-membrane domains that were mainly recycled to the secretory pathway through the Golgi apparatus and a second mainly degradative pathway involving plasma membrane retrieval at the tip. In vivo time-lapse experiments using FM4-64 combined with quantitative analysis confirmed the existence of distinct internalization regions. Ikarugamycin, an inhibitor of clathrin-dependent endocytosis, allowed us to further dissect the endocytic process: electron microscopy and time-lapse studies suggested that clathrin-dependent endocytosis occurs in the tip and subapical regions, because recycling of positively charged nanogold to the Golgi bodies and the consignment of negatively charged nanogold to vacuoles were affected. However, intact positively charged-nanogold transport to vacuoles supports the idea that an endocytic pathway that does not require clathrin is also present in pollen tubes. [ABSTRACT FROM AUTHOR]