Subcellular Lipid Droplets in Vanilla Leaf Epidermis and Avocado Mesocarp Are Coated with Oleosins of Distinct Phylogenic Lineages.

Subcellular lipid droplets (LDs) in diverse plant cells and species are coated with stabilizing oleosins of at least five phylogenic lineages and perform different functions. We examined two types of inadequately studied LDs for coated oleosins and their characteristics. The epidermis but not mesophyll of leaves of vanilla (Vanilla planifolia) and most other Asparagales species contained solitary and clustered LDs (<0.5 µm), some previously studied by electron microscopy and speculated to be for cuticle formation. In vanilla leaves, transcripts of oleosins of the U lineage were present in both epidermis and mesophyll, but oleosin occurred only in epidermis. Immuno-confocal laser scanning microscopy revealed that the LDs were coated with oleosins. LDs in isolated fractions did not coalesce, and the fractions contained heterogeneous proteins including oleosins and diverse lipids. These findings reflect the in situ structure and possible functions of the LDs. Fruit mesocarp of avocado (Persea americana) and other Lauraceae species possessed large LDs, which likely function in attracting animals for seed dispersal. They contained transcripts of oleosin of a novel M phylogenic lineage. Each avocado mesocarp fatty cell possessed one to several large LDs (5 to 20 µm) and at their periphery, numerous small LDs (<0.5 µm). Immuno-confocal laser scanning microscopy revealed that oleosin was present mostly on the small LDs. LDs in isolated fractions coalesced rapidly, and the fraction contained oleosin and several other proteins and triacylglycerols as the main lipids. These two new types of oleosin-LDs exemplify the evolutionary plasticity of oleosins-LDs in generating novel functions in diverse cell types and species.

ROS-induced oxidative stress and apoptosis-like event directly affect the cell viability of cryopreserved embryogenic callus in Agapanthus praecox.

KEY MESSAGE: Oxidative stress and apoptosis-like programmed cell death, induced in part by H 2 O 2 , are two key factors that damage cells during plant cryopreservation. Their inhibition can improve cell viability. We hypothesized that oxidative stress and apoptosis-like event induced by ROS seriously impact plant cell viability during cryopreservation. This study documented changes in cell morphology and ultrastructure, and detected dynamic changes in ROS components (O 2 (·-) , H2O2 and OH·), antioxidant systems, and programmed cell death (PCD) events during embryonic callus cryopreservation of Agapanthus praecox. Plasmolysis, organelle ultrastructure changes, and increases in malondialdehyde (a membrane lipid peroxidation product) suggested that oxidative damage and PCD events occurred at several early cryopreservation steps. PCD events including autophagy, apoptosis-like, and necrosis also occurred at later stages of cryopreservation, and most were apoptosis. H2O2 is the most important ROS molecule mediating oxidative damage and affecting cell viability, and catalase and AsA-GSH cycle are involved in scavenging the intracellular H2O2 and protecting the cells against stress damage in the whole process. Gene expression studies verified changes of antioxidant system and PCD-related genes at the main steps of the cryopreservation process that correlated with improved cell viability. Reducing oxidative stress or inhibition of apoptosis-like event by deactivating proteases improved cryopreserved cell viability from 49.14 to 86.85 % and 89.91 %, respectively. These results verify our model of ROS-induced oxidative stress and apoptosis-like event in plant cryopreservation. This study provided a novel insight into cell stress response mechanisms in cryopreservation.

Evaluations of the mutagenicity of a pigment extract from bulb culture of Hippeastrum reticulatum.

The use of anthocyanins in food products as colorants has been limited because of their instability toward alkaline pH and high temperature. This study aimed to determine color stability and mutagenicity of the anthocyanin-based pigment extract from bulb cultures of Hippeastrum (Hippeastrum reticulatum). The pigment extract retained its reddish-orange color under alkaline conditions (⩽pH 11) and was stable up to 6 h at 95 °C. The mutagenicity of the extract was evaluated in vitro and in vivo. Hippeastrum pigment extract up to 1.25 mg plate(-1) was found non-mutagenic in Ames test using Salmonella typhimurium strain TA98 and TA100. Chromosome aberrations were observed when human lymphocytes were treated with the extract up to 1.5 mg ml(-1). However, the extract up to 1.4 mg ml(-1) was found to exhibit relatively low or no mutagenicity in in vitro comet assays with human lymphocytes. In in vivo micronucleated reticulocyte assay, mice were treated orally with the extract up to 1 g kg(-1). No significant increase of the percentage of micronucleated peripheral reticulocytes compared to the negative control groups was found. Taken together, our study indicates that Hippeastrum pigment extract is potentially applicable as an additive colorant in the diet and related products.

Chromosome diversity and evolution in tribe Lilieae (Liliaceae) with emphasis on Chinese species.

In this paper, karyotype data of the tribe Lilieae in China were analyzed and been superimposed onto a phylogenetic framework constructed by the internal transcribed spacer to investigate the karyotype evolution. Ten parameters for analyzing karyotype asymmetry were assessed and karyotypic idiogram of five genera of Lilieae were illustrated. The results showed that, the relationship of genera in Lilieae that inferred from Maximum Parsimony criteria and Bayesian Inference were congruent with previous studies, which focused on higher level of Liliales. The karyotype showed distinctive among genera, mainly expressed on the location and amount of secondary constrictions and intercalary satellites: the genus Notholirion have neither of them, and the genera Cardiocrinum and Fritillaria have the secondary constriction alone; the genera Lilium and Nomocharis showed both features, and the distribute pattern of the intercalary satellites showed similarity among related clades. The asymmetry that assessed by several methods indicated that the evolution trend of Lilieae did not follow a single direction, but different in each genus. On the sectional level of the genus Lilium (including Nomocharis) the karyotype evolution included three major periods. Combining the chromosomal structure variations and karyotype asymmetry, the chromosome diversity and evolution in Lilieae were quite clear in the light of molecular inference.

An overview on natural cholinesterase inhibitors--a multi-targeted drug class--and their mass production.

Cholinesterase enzyme family consisting of acetylcholinesterase (AChE) and butrylcholinesterase (BChE) is important in pathogenesis of Alzheimer's disease (AD), explained by "cholinergic hypothesis". Accordingly, deficiency of the neuromediator called "acetylcholine" excessive amount of BChE has been well-described in the brains of AD patients. Consequently, cholinesterase inhibition has become one of the most-prescribed treatment strategies for AD. In fact, cholinesterase inhibitors have been also reported for their effectiveness in some other diseases including glaucoma, myasthenia gravies, as well as Down syndrome, lately. They play a role in the action of mechanism of insecticidal drugs such as carbamate derivatives as well as nerve gases such as malathion and parathion. All these utilizations can make them a multi-targeted drug class putting a special emphasis on AD therapy in the first place. Several inhibitors of cholinesterases with synthetic and natural origins are available in drug market; however, the reasons including side effects, relatively low bioavailability, etc. limit their uses in medicine and there is still a great demand to discover new cholinesterase inhibitors. Galanthamine, an alkaloid derivative isolated from snowdrop (Galanthus nivalis L.), is the latest anticholinesterase drug used against AD. Huperzine A, isolated from Huperzia serrata (Thunb.) Trev. is the most-promising drug candidate with potent anticholinesterase effect and it is a licensed anti-AD drug in China. In this review, a short introduction will be given on known cholinesterase inhibitors and, then, galanthamine and huperzine A will be covered in regard with their cholinesterase inhibitory potentials and mass productions by organic synthesis and in vitro culture techniques.

Digital image inpainting and microscopy imaging.

A considerable amount of image processing techniques known as inpainting techniques have been recently developed aiming to provide solutions for filling in missing or damaged regions in a digital image. Typical such techniques reconstruct a defined area by using information from its neighborhood, for example, by completing inside the missing region the isophote lines arriving at its boundaries. In this article, we show that inpainting techniques have considerable potential usefulness in microscopy imaging, even though experimenting and using them in this domain has been almost entirely neglected up until now. In this purpose, we experiment the "curvature-preserving" partial differential equations as a solution to inpainting regions in images collected by several optical and scanning probe microscopy techniques. The results achieved are presented along with a discussion on typical problematic scenarios of microscopy imaging for which this type of techniques can provide a viable solution.

Finite element model of polar growth in pollen tubes.

Cellular protuberance formation in walled cells requires the local deformation of the wall and its polar expansion. In many cells, protuberance elongation proceeds by tip growth, a growth mechanism shared by pollen tubes, root hairs, and fungal hyphae. We established a biomechanical model of tip growth in walled cells using the finite element technique. We aimed to identify the requirements for spatial distribution of mechanical properties in the cell wall that would allow the generation of cellular shapes that agree with experimental observations. We based our structural model on the parameterized description of a tip-growing cell that allows the manipulation of cell size, shape, cell wall thickness, and local mechanical properties. The mechanical load was applied in the form of hydrostatic pressure. We used two validation methods to compare different simulations based on cellular shape and the displacement of surface markers. We compared the resulting optimal distribution of cell mechanical properties with the spatial distribution of biochemical cell wall components in pollen tubes and found remarkable agreement between the gradient in mechanical properties and the distribution of deesterified pectin. Use of the finite element method for the modeling of nonuniform growth events in walled cells opens future perspectives for its application to complex cellular morphogenesis in plants.

Calreticulin expression and localization in plant cells during pollen-pistil interactions.

In this report, the distributions of calreticulin (CRT) and its transcripts in Haemanthus pollen, pollen tubes, and somatic cells of the hollow pistil were studied. Immunoblot analysis of protein extracts from mature anthers, dry and germinated pollen, growing pollen tubes, and unpollinated/pollinated pistils revealed a strong expression of CRT. Both in vitro and in situ studies confirmed the presence of CRT mRNA and protein in pollen/pollen tubes and somatic cells of the pistil transmitting tract. The co-localization of these molecules in ER of these cells suggests that the rough ER is a site of CRT translation. In the pistil, accumulation of the protein in pollen tubes, transmitting tract epidermis (tte), and micropylar cells of the ovule (mc) was correlated with the increased level of exchangeable calcium. Therefore, CRT as a Ca(2+)-binding/buffering protein, may be involved in mechanism of regulation calcium homeostasis in these cells. The functional role of the protein in pollen-pistil interactions, apart from its postulated function in cellular Ca(2+) homeostasis, is discussed.

Carbon dioxide enrichment does not reduce leaf longevity or alter accumulation of carbon reserves in the woodland spring ephemeral Erythronium americanum.

BACKGROUND AND AIMS: Woodland spring ephemerals exhibit a relatively short epigeous growth period prior to canopy closure. However, it has been suggested that leaf senescence is induced by a reduction in the carbohydrate sink demand, rather than by changes in light availability. To ascertain whether a potentially higher net carbon (C) assimilation rate could shorten leaf lifespan due to an accelerated rate of storage, Erythronium americanum plants were grown under ambient (400 ppm) and elevated (1100 ppm) CO2 concentrations. METHODS: During this growth-chamber experiment, plant biomass, bulb starch concentration and cell size, leaf phenology, gas exchange rates and nutrient concentrations were monitored. KEY RESULTS: Plants grown at 1100 ppm CO2 had greater net C assimilation rates than those grown at 400 ppm CO2. However, plant size, final bulb mass, bulb filling rate and timing of leaf senescence did not differ. CONCLUSIONS: Erythronium americanum fixed more C under elevated than under ambient CO2 conditions, but produced plants of similar size. The similar bulb growth rates under both CO2 concentrations suggest that the bulb filling rate is dependant on bulb cell elongation rate, rather than on C availability. Elevated CO2 stimulated leaf and bulb respiratory rates; this might reduce feed-back inhibition of photosynthesis and avoid inducing premature leaf senescence.

[Cytogenetic variation of Ungernia victoris cell lines during cultivation on culture media of different composition].

Chromosome numbers of U. victoris cell lines obtained from the same bulb and cultured for a long time on different agar-solidified and liquid nutrient media differed significantly. The components of the nutrient media including phytohormones did not influence the ratio of cells with different ploidy levels in various lines while transfer of the calluses to the liquid media resulted in the increase of diploid metaphase frequencies.

Isolation, characterization, sequencing and crystal structure of charybdin, a type 1 ribosome-inactivating protein from Charybdis maritima agg.

A novel, type 1 ribosome-inactivating protein designated charybdin was isolated from bulbs of Charybdis maritima agg. The protein, consisting of a single polypeptide chain with a molecular mass of 29 kDa, inhibited translation in rabbit reticulocytes with an IC50 of 27.2 nm. Plant genomic DNA extracted from the bulb was amplified by PCR between primers based on the N-terminal and C-terminal sequence of the protein from dissolved crystals. The complete mature protein sequence was derived by partial DNA sequencing and terminal protein sequencing, and was confirmed by high-resolution crystal structure analysis. The protein contains Val at position 79 instead of the conserved Tyr residue of the ribosome-inactivating proteins known to date. To our knowledge, this is the first observation of a natural substitution of a catalytic residue at the active site of a natural ribosome-inactivating protein. This substitution in the active site may be responsible for the relatively low in vitro translation inhibitory effect compared with other ribosome-inactivating proteins. Single crystals were grown in the cold room from PEG6000 solutions. Diffraction data collected to 1.6 A resolution were used to determine the protein structure by the molecular replacement method. The fold of the protein comprises two structural domains: an alpha + beta N-terminal domain (residues 4-190) and a mainly alpha-helical C-terminal domain (residues 191-257). The active site is located in the interface between the two domains and comprises residues Val79, Tyr117, Glu167 and Arg170.

Programmed cell death progressively models the development of anther sporophytic tissues from the tapetum and is triggered in pollen grains during maturation.

To characterize the spatial and temporal occurrence of programmed cell death (PCD) in Lilium anther tissues, we used both microscopical and molecular markers of apoptosis for developmental stages from meiosis to pollen release. The first hallmarks of PCD include cell condensation and shrinkage of the cytoplasm, separation of chromatin into delineated masses, and DNA fragmentation in the tapetum as early as the premeiosis stage. PCD then extended to other anther sporophytic tissues, leading to anther dehiscence. Although the PCD clearly affected the endothecium and the epidermis, these two cell layers remained alive until anther dehiscence. In pollen, no sign of PCD was found until pollen mitosis I, after what apoptotic features developed progressively in the vegetative cell. In addition, DNA ladders were detected in all sporophytic tissues and cell types throughout pollen development, whereas in the male gametophyte DNA ladders were only detected during pollen maturation. Our data suggest that PCD is a progressive and active process affecting all the anther tissues, first being triggered in the tapetum.

[Contractile proteins in chemical signal transduction in plant microspores].

Involvement of contractile components in chemical signal transduction from the cell surface to the organelles was studied using unicellular systems. Neurotransmitters dopamine and serotonin as well as active forms of oxygen hydrogen peroxide and tert-butyl peroxide were used as chemical signals. Experiments were carried out on vegetative microspores of field horsetail Equisetum arvense and generative microspores (pollen) of amaryllis Hippeastrum hybridum treated with cytochalasin B (an inhibitor of actin polymerization in microfilaments), colchicine, and vinblastine (inhibitors of tubulin polymerization in microtubules). Both types of thus treated microspores demonstrated suppressed development, particularly, for cytochalasin B treatment. At the same time, an increased typical blue fluorescence of certain cell regions (along the cell wall and around nuclei and chloroplasts) where the corresponding contractile proteins could reside was observed. In contrast to anticontractile agents, dopamine, serotonin B, and the peroxides stimulated microspore germination. Microspore pretreatment with cytochalasin B and colchicine followed by the treatment with serotonin, dopamine, or the peroxides decreased the germination rate. Involvement of actin and tubulin in chemical signal transduction from the cell surface to the nucleus is proposed.

An evolutionary change in telomere sequence motif within the plant section Asparagales had significance for telomere nucleoprotein complexes.

In association with a phylogenetic tree of Asparagales, our previous results showed that a distinct clade included plant species where the ancestral, Arabidopsis-type of telomeric repeats (TTTAGGG)n had been partially, or fully, replaced by the human-type telomeric sequence (TTAGGG)n. Telomerases of these species synthesize human repeats with a high error rate in vitro. Here we further characterize the structure of telomeres in these plants by analyzing the overall arrangement of major and minor variants of telomeric repeats using fluorescence in situ hybridization on extended DNA strand(s). Whilst the telomeric array is predominantly composed of the human variant of the repeat, the ancestral, Arabidopsis-type of telomeric repeats was ubiquitously observed at one of the ends and/or at intercalary positions of extended telomeric DNAs. Another variant of the repeat typical of Tetrahymena was observed interspersed in about 20% of telomerics. Micrococcal nuclease digestions indicated that Asparagales plants with a human-type of telomere have telomeric DNA organised into nucleosomes. However, unexpectedly, the periodicity of the nucleosomes is not significantly shorter than bulk chromatin as is typical of telomeric chromatin. Using electrophoretic mobility shift assays we detected in Asparagales plants with a human type of telomere a 40-kDa protein that forms complexes with both Arabidopsis- and human-type G-rich telomeric strands. However, the protein shows a higher affinity to the ancestral Arabidopsis-type sequence. Two further proteins were found, a 25-kDa protein that binds specifically to the ancestral sequence and a 15-kDa protein that binds to the human-type telomeric repeat. We discuss how the organisation of the telomere repeats in Asparagales may have arisen and stabilised the new telomere at the point of mutation.

[Reactive oxygen forms and luminescence of intact microspore cells]. / Aktivnye formy kisloroda i liuminestsentsiia intaktnykh kletok mikrospor.

The participation of reactive oxygen species (ROS) in luminescence (chemiluminescence and autofluorescence induced by ultraviolet light of 360-380 nm) was analyzed. Microspores, the pollen (male gametophyte) of Hippeastrum hybridum, Philadelphus grandiflorus, and Betula verrucosa and vegetative microspores of the spore-breeding plant Equisetum arvense served as models. It was found that the addition of the chemiluminescent probe lucigenin, which luminesces in the presence of superoxide anionradicals, leads to intensive chemiluminescence of microspores. No emission was observed in the absence of lucigenin and in the presence of the dye luminol as a chemiluminescent probe. The emission decreased significantly if superoxide dismutase, an enzyme of the superoxide anionradical dismutation during which this radical disappeared, was added before the dye addition. The autofluorescence intensity of microspores decreased in the presence of both superoxide dismutase and peroxidase, an enzyme destroying hydrogen peroxide and organic peroxides. The most significant effect was noted after the addition of peroxidase, which indicates a greater contribution of peroxides to this type of emission. The fumigation with ozone, which increases the amount of ROS on the cell surface, enhanced the intensity of the chemiluminescence of microspores with lucigenin, but decreased the intensity of the autofluorescence of microspores. Exogenous peroxides (hydrogen peroxide and tert-butylhydroperoxide) stimulated the autofluorescence of pollen and vegetative spores in a concentration-dependent manner. It was shown that the formation of ROS contributes to the luminescence of plant microspores, which reflects their functional state.

Homoisoflavonoids and xanthones from the tubers of wild and in vitro regenerated Ledebouria graminifolia and cytotoxic activities of some of the homoisoflavonoids.

Eleven homoisoflavonoids and two xanthones were isolated and characterized from the bulbs of Ledebouria graminifolia. Five of the homoisoflavonoids are new compounds and were identified as: 5-hydroxy-7-methoxy-3-(4'-hydroxybenzyl)-4-chromanone, 5-hydroxy-6,7-dimethoxy-3-(4'-hydroxybenzyl)-4-chromanone, 5,7,8-trimethoxy-3-(4'-hydroxybenzyl)-4-chromanone, 5-hydroxy-3',4',7-trimethoxyspiro[2H-1-benzopyran-7'-bicyclo[4.2.0]octa-trien]-4-one, 5,7-dihydroxy-3',4'-dimethoxyspiro[2H-1-benzopyran-7'-bicyclo[4.2.0]octa-trien]-4-one. Structures were elucidated by extensive 1D, and 2D NMR spectroscopy and HRMS. A method for tissue culture was developed and the bulbs of mature plants were found to contain all the compounds isolated from the wild specimens of L. graminifolia.

Organization of cell walls in Sandersonia aurantiaca floral tissue.

Visual symptoms of the onset of senescence in Sandersonia aurantiaca flowers begin with fading of flower colour and wilting of the tissue. When fully senescent, the flowers form a papery shell that remains attached to the plant. The cell walls of these flowers have been examined to determine whether there are wall modifications associated with the late stages of expansion and subsequent senescence-related wilting. Changes in the average molecular size of pectin were limited through flower opening and senescence, although there was a loss of neutral sugar-containing side-branches from pectins in opening flowers, and the total amounts of pectin and cellulose continued to rise in cell walls of fully senescent sandersonia flowers. Xyloglucan endotransglycosylase activity increased in opening and mature flowers, but declined sharply as flowers wilted. Concomitantly, the proportion of hemicellulose polymers of increasing molecular weight increased from flower expansion up to the point at which wilting occurred. Approximately 50% of the non-cellulosic neutral sugar in mature flower cell walls was galactose, primarily located in an insoluble cell wall fraction. Total galactose in this fraction increased per flower with maturity, then declined at the onset of wilting. Beta-galactosidase activity was low in expanding tepals, but increased as flowers matured and wilted.

Pectin secretion and distribution in the anther during pollen development in Lilium.

Using the monoclonal antibodies JIM 5 and 7, pectin was immunolocalized and quantitatively assayed in three anther compartments of Lilium hybrida during pollen development. Pectin levels in both the anther wall and the loculus increased following meiosis, were maximal during the early microspore stages and declined during the remainder of pollen ontogenesis. In the microspores/pollen grains, pectin was detectable at low levels during the microspore stages but accumulated significantly during pollen maturation. During early microspore vacuolation, esterified pectin epitopes were detected both in the tapetum cytoplasm and vacuoles. In the anther loculus, the same epitopes were located simultaneously in undulations of the plasma membrane and in the locular fluid. At the end of microspore vacuolation, esterified pectin epitopes were present within the lipids of the pollenkitt, and released in the loculus at pollen mitosis. Unesterified pectin epitopes were hardly detectable in the cytoplasm of the young microspore but were as abundant in the primexine matrix as in the loculus. During pollen maturation, both unesterified and esterified pectin labelling accumulated in the cytoplasm of the vegetative cell, concurrently with starch degradation. In the mature pollen grain, unesterified pectin epitopes were located in the proximal intine whereas esterified pectin epitopes were deposited in the distal intine. These data suggest that during early microspore development, the tapetum secretes pectin, which is transferred to the primexine matrix via the locular fluid. Further, pectin is demonstrated to constitute a significant component of the pollen carbohydrate reserves in the mature grain of Lilium.

Cytochemical evidence for the presence of actin in the nucleus of the voodoo lily appendix.

Immunoflorescence microscopy of sections of the voodoo lily Sauromatum guttatum appendix stained with monoclonal antibodies against alpha-smooth muscle actin and cytoplasmic actin revealed different staining intensity of different parts of the cell. The anti-cytoplasmic-actin recognized antigens present mainly in the cytoplasm, and the anti-alpha-smooth muscle-actin recognized more intensively antigens present in the nuclei. A positive staining of the nucleus was also obtained with FITC-phalloidin confirming the presence of actin in its filamenous form in the nucleus. The presence of a nuclear alpha-smooth muscle-actin-like protein was further confirmed by confocal laser microscopy. On Western blots, the two anti-actins labelled a protein band that comigrated with standard actin at the approximate molecular weight of 43 kDa. Several other proteins interacted with the two antibodies to a different degree. The monoclonal antibodies against beta-tubulin subunit stained only the periphery of the cytoplasm and anti-pan cytoplasmic myosin stained the cytoplasm weakly. On a Western blot, anti-beta-tubulin subunit primarily recognized a protein band at the appropriate molecular weight of 50 kDa. This is the first cytochemical evidence for the presence of alpha-smooth muscle-actin-like protein in the plant nucleus.

Adhesion and cell movement during pollination: cherchez la femme.

Pollination involves an interaction between the female tissues (stigma, style and ovary) and the male gametophyte or the pollen tube cell, which contains the sperm cells. Freezing methods now allow us to visualize the extracellular matrices that guide pollen tubes to the ovary. Adhesion of the pollen tube to these specialized extracellular matrices might be a mechanism of guidance and tube cell movement in the style. In lily, the stylar adhesion molecules are a pectin and a small, basic cysteine-rich protein, both of which are necessary to induce tube cell adhesion to an artificial, in vitro style matrix.