Protein-storing vacuoles in inner bark and leaves of softwoods

Summary The seasonal occurrence of protein-storage vacuoles in parenchyma cells of the inner bark and leaf tissues of seven softwood species was examined. Previously published results showed that these organelles often fill the phloem parenchyma cells of the inner bark tissues in overwintering hardwoods, whereas they are absent from this tissue during the summer. We hypothesize that the organelles are involved in the storage of reduced nitrogen during wintering, in a manner analogous to protein bodies of seeds. A survey of the phloem and cambial parenchyma tissues in six evergreen softwood species (Pinus strobus, P. sylvestris, Picea abies, P. glauca, Abies balsamea, and Thuja occidentalis) and in one deciduous softwood species (Larix decidua) was conducted. There was a large variation in the degree and timing of protein-storage vacuole formation between the individual genera and species. The organelles were not seen in summer samples of inner bark tissues of any of the genera or species examined. Protein-storage vacuoles were common in the bark tissues of Pinus, Abies and Thuja, occasionally seen in Picea, and rarely found in Larix during the winter. One-year-old leaves were also examined, since in all but Larix they are overwintering structures and can act as potential sites of nitrogen storage. Protein-storage vacuoles were present in Pinus and Thuja leaf tissue in both summer and winter, in Abies during winter only, and were absent from Picea leaf tissue at all times. These results indicate that the formation of protein-storage vacuoles prior to overwintering is not a ubiquitous phenomenon in softwoods.     

The 32-Kilodalton Vegetative Storage Protein of Salix microstachya Turz : Characterization and Immunolocalization

A 32-kilodalton vegetative storage protein, found in Salix microstachya Turz. bark during the overwintering period, was purified and characterized using several polyacrylamide gel electrophoretic procedures. Solubility characteristics and amino acid analyses were also performed. The protein is water soluble, is glycosylated, has no disulfide-bonded subunits, but is composed of a family of isoelectric isomers. The majority of these isomers are basic. Characteristic of storage proteins, the protein is rich in glutamine/glutamate and asparagine/aspartate (28%), the basic nature of the isomers indicating that most of these amino acid residues are in the amide form. The protein was purified using preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and antibodies raised in chickens. Immunoblot analysis suggested an annual cyclic nature of the accumulation and mobilization of this vegetative storage protein. Immunologically, it is related to a similar molecular weight protein found in the bark of Populus deltoides Marsh. but not to any overwintering storage proteins of the other hardwoods tested. Indirect immunolocalization revealed that the protein was sequestered in protein-storage vacuoles in parenchymatous cells of the inner bark tissues of Salix during the winter months.     

Proteins as a potential nitrogen storage compound in bark and leaves of several softwoods

Summary The occurrence of vegetative storage proteins in the leaf and bark tissues of several softwood species during overwintering was investigated by sodium dodecyl sulphate polyacrylamide electrophoresis. Monthly protein profiles from leaves and bark of six evergreen softwood species (Pinus strobus, P. sylvestris, Picea abies, P. glauca, Abies balsamea, and Thuja occidentalis) and the bark of one deciduous softwood species (Larix decidua) suggest that storage proteins are present in bark tissues of L. decidua, Pinus sylvestris, and P. strobus. The remaining species did not show similar specific proteins. However, the total soluble protein content which was determined during active growth and during overwintering in the same tissues indicated that protein levels were higher in the winter compared to the summer in the bark of all species and in the leaves of Pinus spp. and T. occidentalis. While vegetative storage proteins do not appear prevalent in all softwood species, proteins may constitute a major form of overwintering nitrogen storage for many species.     

Sambucus nigra agglutinin is located in protein bodies in the phloem parenchyma of the bark

The bark of some young woody stems contains storage proteins which are subject to an annual rhythm: they accumulate in the autumn and are mobilized in the spring. We show here that the bark phoem-parenchyma cells of Sambucus nigra L. contain numerous protein bodies, and that the bark lectin (S. nigra agglutinin) which undergoes an annual rhythm is localized in these protein bodies. The protein bodies in the cotyledons of legume seeds also contain lectin, indicating that lectins may be storage compounds themselves or may have a function in storage and-or mobilization processes.Abbreviations PBS phosphate-buffered saline - IgG immunoglobulin - SNA Sambucus nigra agglutinin     

Vacuole proteins in parenchyma cells of secondary phloem and xylem of Dalbergia odorifera

Summary Light- and electron-microscopic observations were made on the stem parenchyma cells of Dalbergia odorifera T. Chen (Papilionaceae), a tropical deciduous tree. In the secondary phloem of branchlet and trunk, all of the parenchyma cells except companion cells contain vacuole proteins. Only the outer secondary xylem of branchlets, but not trunk secondary xylem, has proteins in the ray parenchyma and the vasicentric parenchyma. The xylem vacuole proteins begin to accumulate at the end of the growing period and they disappear after the first flush of growth in spring. The vacuole proteins in phloem cells, particularly in the cells near the cambium, also show seasonal fluctuations. Under the electron microscope, the vacuole proteins appear as fibrous materials in aggregation or in more or less even dispersion, and they occur in the large central vacuoles during both the growth and dormant periods. According to the published studies, the stem storage proteins in the temperate trees appear as small protein-storage vacuoles or protein bodies, and the proteins in the tropical trees occur in large central vacuoles. This distinction is assumed to be related to the differences in the nature of dormancy between temperate and tropical trees.     

A survey of seasonal bark proteins in eight temperate hardwoods

Summary Bark proteins of eight temperate hardwoods were analyzed by SDS-PAGE at monthly intervals to determine whether an accumulation of specific proteins, potential storage proteins, occurred in the fall at the time of leaf senescence. Storage proteins were identified as proteins that accumulated during the fall and were present in reduced amounts in the summer. Total protein levels were higher in the winter than in the summer in Fagus sylvatica, Fraxinus americana, Tilia americana, Alnus glulinosa, Betula papyrifera and Querus rubra, but not in Gleditsia triacanthos or Robinia pseudoacacia. Betula contained the most abundant storage protein, although in all species minor bands, which fluctuated seasonally, could be identified. With the exception of Alnus and Betula, results generally correlated with previous microscopy studies of these tree species, which showed varying amounts of protein storage vacuoles present in phloem parenchyma cells during the winter, but not during the summer.     

Seasonal fluctuation of dehydrins is related to osmotic status in Scots pine needles

Seasonal variation in dehydrins and other soluble proteins of Scots pine (Pinus sylvestris L.) needles, buds and bark were analyzed monthly for 1 year from 1998 to 1999. Dehydrin-related proteins of 60 and 56 kDa were identified immunologically in all tissues. The concentration of the 60-kDa dehydrin was highest during the winter (October-February) in buds and bark but increased in early spring (March-May) in needles. Accumulation of the 60-kDa dehydrin in the needles in springtime was related to the decreasing osmotic potentials of the needles. The 56-kDa dehydrin was present only during the growing season, as was a 50-kDa dehydrin, which only appeared in bud and bark tissues. The soluble protein concentration of needles did not differ significantly between seasons, but in bark and bud tissues the protein concentrations were at their lowest level in newly grown tissues (June-August). The level of several polypeptides was higher during the winter-spring period than in the growing season, especially in bark and bud tissues. These proteins may be related to cold hardiness or dormancy in overwintering Scots pine. Dehydrin-related proteins in needles are linked to springtime changes in the osmotic status of needles rather than to their cold acclimation.     

Proteins in the roots of the perennial weeds chicory (Cichorium intybus L.) and dandelion (Taraxacum officinale Weber) are associated with overwintering

Roots are the overwintering structures of herbaceous perennial weeds growing in temperate climates. During the fall they accumulated reserves which are remobilized when growth resumes in the spring. An 18kDa (kilodalton) protein increases in both chicory and dandelion roots during the fall months. The proteins in both species are antigenically similar, and are recognized also by an antibody to a storage-protein deposited in Jerusalem artichoke (Helianthus tuberosus) tubers. In chicory, the protein is root-specific, but in dandelion it is detectable in the flowers, vestigial stem and the seed. Electrophoretic characterization of the 18-kDa protein shows that it is a single polypeptide, without subunits, with charge isomers of pI values close to pH 6.5. The major protein present in chicory and dandelion roots is unlike the vegetative storage proteins recently found in soybean or the storage proteins in the bark of trees.     

Studies on the Fine Structure of the Scutellum of the Oat Seed During Germination

Structural changes in the seutellar parenchyma and epithelial cells of oats during the first 3 days of germination were followed by electron microscopy. The seutellar parenchyma cells contain more protein bodies than the epithelial cells, otherwise the general fine structures of the two types of cells arc quite similar: When the seed starts to germinate the protein bodies change into vacuoles and the proteins inside the protein bodies gradually disappear. Spherosomes are in abundance ill the seutcllar cells of the dry seed. Few disappeared during germination. Other cellular organelles, such as the mitochondria, endoplasmie reticulum, plastids, Golgi apparatus and glyoxysomes are scarcely seen in the seutellar cells of the dry seed. They become more obvious and easily recognizable after germination. In the dry seed, the walls of the epithelial cell that abut the endospernl show a two layered structure, consisted of an inner and outer layer. The outer layer becomes hydrolysed during seed germination, but the inner layer remains intact. The scutetlar epithelial cells are known for their ability to secret enzymes ute and absorb nutrients from the endosperm. But in the fine structural studies we have not been able to locate any specific strurcture that could be related to their known functions of enzyme secretion and nutrient absorption.     

Histochemical and ultrastructural changes in the haustorium of date (Phoenix dactylifera L.)

Summary During imbibition ofPhoenix dactylifera embryos, all cotyledon cells show the same changes: protein and lipid bodies degrade, smooth endoplasmic reticulum (ER) increases in amount, and dictyosomes appear. At germination, the distal portion of the cotyledon expands to form the haustorium. At this time, epithelial cells have a dense cytoplasm with many extremely small vacuoles. Many ribosomes are present along with ER, dictyosomes, and mitochondria. The parenchyma cells have large vacuoles and a small amount of peripheral cytoplasm. Between 2 and 6 weeks after germination, epithelial cells still retain the dense cytoplasm and many organelles appear: glyoxysomes, large lipid bodies, amyloplasts, large osmiophilic bodies, and abundant rough and smooth ER which appear to merge into the plasmalemma. A thin electron-transparent inner wall layer with many small internal projections is added to the cell walls. Starch grains appear first in the subsurface and internal parenchyma and subsequently in the epithelium. Lipid bodies, glyoxysomes, protein, and osmiophilic bodies occur in the epithelial and subepithelial cell layers but not in the internal parenchyma. At 8 weeks after germination, the cytoplasm becomes electron transparent, vacuolation occurs, lipid bodies and osmiophilic bodies degrade, and the endomembranes disassemble. After 10 weeks, the cells are empty. These data support the hypothesis that the major functions of the haustorium are absorption and storage.     

ULTRASTRUCTURAL CHANGES ASSOCIATED WITH UTILIZATION OF METABOLITE RESERVES AND TRICHOME DIFFERENTIATION IN THE PROTOCORM OF VANDA

Certain aspects of protocorm development in Vanda were examined ultrastructurally. The parenchymal cells of the protocorm accumulate substantial quantities of lipid, protein, and carbohydrate reserves which disappear gradually with the senescence of the parenchymatous region. The proteinaceous reserves appear initially as discrete bodies which become intimately associated with clusters of small tubules. The tubules eventually disperse throughout the cytoplasm and disappear following depletion of the protein bodies. The lipid reserves also appear as discrete bodies and are associated with an electron dense, laminated inclusion which appears to increase in size with the disappearance of the lipid bodies. While plastids in the meristematic cells differentiate a well-developed thylakoid system and contain little starch, those of the parenchymal cells contain large starch grains and numerous osmiophilic droplets and develop meager thylakoid systems. Membrane-bound crystalline structures of hexagonal and rhomboid cross section occur frequently in the cytoplasm of senescent parenchyma cells. Trichome initials, which differentiate from the epidermis, contain few conventional organelles and exhibit numerous membrane-bound structures containing many small crystalline inclusions. Numerous vesicles accumulate at the tips of the trichomes in spaces between the cell wall and the plasmalemma.     

Enigmatic adult overwintering in damselflies: coexistence as weaker intraguild competitors due to niche separation in time

Odonata, like most freshwater invertebrates, tend to overwinter in water due to the thermal properties of a water environment. Winter damselflies (genus Sympecma), however, hibernate as adults in terrestrial habitats. The strategy of adult overwintering combined with high mortality is associated with several unique adaptations to semiarid conditions, but winter damselflies maintain this unique life history throughout almost the entire Palaearctic. We assume that the unique strategy of adult overwintering in temperate zones is indirectly maintained by niche separation in time. We used phenological data from the Czech Republic to compare the seasonal phenology of Sympecma spp. with other coexisting odonate species. Seasonal population growth patterns between S. fusca and other coexisting species representing different life histories were compared using GLMMs and LME. The models showed negative non-linear dependence between the population growth of S. fusca and the estimated abundance of compared species. We found that the specific strategy of adult overwintering makes it possible to avoid seasonal maxima of competition and predation in adult and larval stages. Adults may benefit from free niches during spring while larvae may benefit from size advantage among intraguild competitors and optimal conditions for development.     

Ethylene in induced conifer defense: cDNA cloning, protein expression, and cellular and subcellular localization of 1-aminocyclopropane-1-carboxylate oxidase in resin duct and phenolic parenchyma cells

Members of the Pinaceae family have complex chemical defense strategies. Conifer defenses associated with specialized cell types of the bark involve constitutive and inducible accumulation of phenolic compounds in polyphenolic phloem parenchyma cells and oleoresin terpenoids in resin ducts. These defenses can protect trees against insect herbivory and fungal colonization. The phytohormone ethylene has been shown to induce the same anatomical and cellular defense responses that occur following insect feeding, mechanical wounding, or fungal inoculation in Douglas fir (Pseudotsuga menziesii) stems (Hudgins and Franceschi in Plant Physiol 135:2134–2149, 2004). However, very little is known about the genes involved in ethylene formation in conifer defense or about the temporal and spatial patterns of their protein expression. The enzyme 1-aminocyclopropane-1-carboxylate oxidase (ACO) catalyzes the final step in ethylene biosynthesis. We cloned full-length and near full-length ACO cDNAs from three conifer species, Sitka spruce (Picea sitchensis), white spruce (P. glauca), and Douglas fir, each with high similarity to Arabidopsis thaliana ACO proteins. Using an Arabidopsis anti-ACO antibody we determined that ACO is constitutively expressed in Douglas fir stem tissues and is up-regulated by mechanical wounding, consistent with the wound-induced increase of ethylene levels. Immunolocalization showed cytosolic ACO is predominantly present in specialized cell types of the wound-induced bark, specifically in epithelial cells of terpenoid-producing cortical resin ducts, in polyphenolic phloem parenchyma cells, and in ray parenchyma cells.J.W. Hudgins and Steven G. Ralph contributed equally to this work.     

QUANTITATIVE ULTRASTRUCTURE AND PROTEIN COMPOSITION OF DATE PALM (PHOENIX DACTYLIFERA) SEEDS: A COMPARATIVE STUDY OF ENDOSPERM VS. EMBRYO

Comparative studies on the ultrastructure and protein composition of the embryo and endosperm of date palm (Phoenix dactylifera L.) were conducted. Cells of the embryo cotyledon and endosperm function in reserve storage and contained cell walls, nuclei, and cytoplasm rich in lipid and protein bodies. Morphometric analysis from light and electron micrographs showed that the cell walls of the endosperm occupied 65% of the total cell volume, but only 6% in the embryo. The protein bodies of the endosperm accounted for 11%, whereas those of the embryo occupied more than half of the total cell volume. The volume of organelles and organelle-free cytoplasm in the endosperm was negligible, suggesting that most of the extractable endosperm proteins are localized in the protein bodies. Extractable proteins in the embryo may come from cytoplasm, protein bodies, and other organelles. The endosperm contains relatively lower amounts of proteins than does the embryo. Proteins extracted from both tissues were compared using SDS-polyacrylamide gel electrophoresis, tube gel isoelectric focusing, and two-dimensional electrophoresis. Proteins of both the tissues were heterogeneous in molecular mass and charge. The majority of the proteins were similar in molecular mass and charge in the two tissues, suggesting that most of the storage proteins are probably the same. However, there were also several embryo- and endosperm-specific proteins apparent in both the first- and second-dimension gels. The endosperm-specific proteins may play an important role in germination and seedling development.     

Embryonic Stem Cells: Spontaneous and Directed Differentiation

The specific structural features of embryonic stem cells and embryoid bodies and mechanisms of their differentiation in different cell types are considered. The mouse embryonic stem cells (line R1) formed multilayer colonies which enlarged as a result of fast cell division. Embryoid bodies that derived from embryonic stem cells consisted of an outer layer, an inner layer, and an internal cavity. The structure of cells of the outer and inner layers markedly differed. Spontaneous and directed differentiation of embryoid bodies is determined by some unspecific and specific factors (growth and differentiation factors and extracellular matrix proteins). Retinoic acid, the most commonly used inducer of differentiation of the embryonic stem cells, induces different types of differentiation when applied at different concentrations. The sequence of expression of tissue specific genes and proteins during differentiation of the embryonic stem cells in vitrois similar to that in vivo.     

Carbohydrate Metabolism in Taproots of Medicago sativa L. during Winter Adaptation and Spring Regrowth

Our objective was to identify amylases that may participate in starch degradation in alfalfa (Medicago sativa L.) taproots during winter hardening and subsequent spring regrowth. Taproots from field-grown plants were sampled at intervals throughout fall, winter, and early spring. In experiment 1, taproots were separated into bark and wood tissues. Concentrations of soluble sugars, starch, and buffer-soluble proteins and activities of endo- and exoamylase were determined. Starch concentrations declined in late fall, whereas concentrations of sucrose increased. Total amylolytic activity (primarily exoamylase) was not consistently associated with starch degradation but followed trends in soluble protein concentration of taproots. This was especially evident in spring when both declined as starch degradation increased and shoot growth resumed. Activity of endoamylase increased during periods of starch degradation, especially in bark tissues. In experiment 2, a low starch line had higher specific activity of taproot amylases. This line depleted its taproot starch by late winter, after which taproot sugar concentrations declined. As in experiment 1, total amylolytic activity declined in spring in both lines, whereas that of endoamylase increased in both lines even though little starch remained in taproots of the low starch line. Several isoforms of both amylases were distinguished using native polyacrylamide electrophoresis, with isoforms being similar in bark and wood tissues. The slowest migrating isoform of endoamylase was most prominent at each sampling. Activity of all endoamylase isoforms increased during winter adaptation and in spring when shoot growth resumed. Endoamylase activity consistently increased at times of starch utilization in alfalfa taproots (hardening, spring regrowth, after defoliation), indicating that it may serve an important role in starch degradation.     

A histological and histochemical study of the cotyledons ofPhaseolus vulgaris L. during germination

Summary The cotyledon ofPhaseolus vulgaris L. comprises four tissues: epidermis, abaxial hypodermis, storage parenchyma, and procambium. A complex intercellular space system is present throughout the storage tissue and comprises about 16% of the cotyledon volume. All the cells contain protein bodies, and the hypodermis and storage parenchyma also contain starch grains. The epidermal cells are at the 2 C level of DNA, those of the hypodermis at the 4 C level, and the storage cells vary from 8 C to 32 C. During germination stomata differentiate in the epidermis. Reserve mobilization begins in the cells furthest from the epidermis and from the vascular tissue. Protein is removed from these cells with little or no coalescence of protein bodies. The DNA content of the nuclei decreases. The cell walls swell and then decrease in thickness as material is removed. Finally the nuclei and cytoplasm disappear and the cells collapse. In the cells near vascular bundles the protein bodies coalesce before losing their protein. The DNA content of the nuclei declines but nuclei and cytoplasm are still present at abscission. These cells do not collapse. Cytoplasmic RNA content is highest near the abaxial surface. Most of the RNA is removed during the first three days of germination.     

THE PRESENCE OF PROTEASE-DIGESTIBLE MATERIAL IN GOLGI VESICLES DURING ENDOSPERM DEVELOPMENT OF SELECTED CEREALS

The presence of dictyosomes secreting densely stained vesicles throughout endosperm protein body formation was confirmed for four cereals (rice, Oryza sativa L.; hard red winter wheat, Triticum aestivum L.; winter feed barley and spring malting barley, Hordeum vulgare L.; oats, Avena sativa L.). The contents of the Golgi vesicles and protein bodies were digested with proteases for all cereals except rice. It was found in the case of rice that OsO₄ altered the proteins in the Golgi apparatus and protein bodies making them resistant to protease digestion. These results imply that the Golgi apparatus plays an important role in the concentration and transport of storage proteins into vacuoles.