The positional differentiation of abscission zones during the development of leaves of Sambucus nigra and the response of the cells to auxin and ethylene

Summary Abscission in the leaf rachis of Sambucus nigra L. is preceded by a positional differentiation of zone cells that enlarge and separate in response to ethylene but not to auxin. These cells are absent from youngest leaves, and such leaves do not abscind even in ethylene; other cells of the immature rachii will enlarge in response to auxin. These two classes of target cells are always recognisable by their opposing responses to auxin and ethylene. Prior to separation zone cells exposed to ethylene show considerable activation of the cytoplasm, many polysomes, elongate endoplasmic reticulum and highly dilated dictyosomes with many associated vesicles. Treatment with auxin precludes these changes, and abscission is always retarded: high levels of ethylene must be added to overcome the auxin inhibition. The differentiation of zone cells and their ethylene-stimulated growth and activation are prerequisites for rachis abscission in Sambucus. Such cell development may be of general occurrence prior to organ abscission in plants.Abbreviation IAA indole-3yl-acetic acid     

Identification of polypeptides specific to rachis abscission zone cells of Sambucus nigra

Specific polypeptides and antigenic determinants in abscission zone cells of the leaf rachis of Sambucus nigra L. (elderberry) were identified. Extracts from abscission zone tissue (OZ) and from ethylene-treated abscission zone tissue after separation (Zone) were probed, using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunological techniques, for unique peptide components absent from neighbouring non-zone mid-rachis tissue (MR). Ouchterlony immuno-diffusion revealed differences in the spectrum of antigenic determinants possessed by each tissue type when challenged with antiserum raised against OZ. Immuno-electrophoresis showed a basic polypeptide which is expressed preferentially in the abscission zone.
Immune-competition of OZ, MR and Zone extracts using immuno-affinity column chromatography has identified polypeptides of ca 34, ca 32, and ca 28 kDa which are (up to the limits of detection) abscission-cell specific. An antibody raised against the ca 34 kDa polypeptide recognises a peptide of ca 34 kDa present in OZ and Zone. This peptide is absent from MR.
These results suggest that the specific positional differentiation of ethylene-responsive target cells which constitute the leaf rachis abscission zone in S. nigra is accompanied by the expression of zone-cell-specific antigenic determinants which are not expressed by non-target neighbouring tissue.     

Biochemical changes in primary wheat leaves during growth and senescence

Changes in the different forms of the total activity of peroxidases (soluble, ionically and covalently bound), in the soluble isoperoxidase composition, and in the content of protein and chlorophyll during growth and senescence of the intact primary leaves of spring wheat were studied. The forms of peroxidases are in inverse relationship to the growth and increase during development and senescence, especially towards the end of senescence. The activity of peroxidases does not follow the physiological age of wheat leaves during the whole period of vegetation unlike the contents of protein and chlorophyll. The intensity of anodic and cathodic peroxidase isoenzyme bands 1 and 5 increases during senescence, but anodic bands number 2 and 4, cathodic bands 2 and 3 decrease. Changes in the activity of peroxidases and in the content of protein and chlorophyll as indicators of senescence are discussed.     

Temporal and spatial expression of mRNAs encoding pathogenesis-related proteins during ethylene-promoted leaflet abscission in Sambucus nigra*

Differential screening of a cDNA library generated from RNA extracted from ethylene-treated leaflet abscission zones of Sambucus nigra resulted in the isolation of 20 abscission-related clones. These clones could be grouped into seven families. Sequencing of members of these families revealed that the majority encoded pathogenesis-related (PR) proteins, and these could be identified by sequence homology as a polyphenol oxidase (PPO), a PR-1 type protein, a Chial type chitinase, a PR-4 type protein similar to the potato win peptides, a PR-6 type proteinase inhibitor, a Chia4 type chitinase and a metallothionein-like protein (Coupe, Taylor & Roberts 1995, Planta 197, 442–447). Northern analysis revealed that these mRNAs were not expressed in freshly excised material but accumulated primarily in the abscission zone tissue after 18 h of exposure to ethylene at a time when abscission of the leaflet explants had reached 70%. Expression of the PPO and the Chia4-type chitinase was ethylene-dependent while that of the PR-4 type was up-regulated in the abscission zone tissue in the absence of the gas. The characterization of these mRNAs and their encoded proteins is presented and their possible roles during abscission are discussed.     

Autoradiographic and ultrastructural study of Cucurbita pepo root cells during their growth and differentiation

Summary Cortex cells of the root meristem of Cucurbita pepo (0.0–0.5 mm from the cap junction), in the 3–4, 5–6 and 7–8 mm segments above the root tip, and the cells of the first three layers of lateral part of root cap were the object of the present study. The volume of cortex cells increases more than 20 times in the 7–8 mm segment as compared with meristematic cells, and the volume of cytoplasm about sevenfold. The largest increment of the cytoplasmic volume occurs between 0.5–6.0 mm. In consecutive root segments the sustained increase of the volume of nuclei takes place. By applying autoradiography the following processess have been investigated: DNA synthesis (³H thymidine uptake), template activity of DNA (³H actinomycin D(³H AMD)-binding), RNA synthesis (³H uridine incorporation), and protein synthesis (³H leucine). In the root cap cells and in segments where meristematic activity is over, DNA is replicated by endomitosis. On the basis of nuclear labelling it appears that nuclei in the 3–4 mm segment reach 4C ploidy state, but in the 7–8 mm segment half of the nuclei reach the 8C ploidy state. Most of the root cap cells are 4C, the remaining cells are 8C. Considering the uptake of ³H thymidine into nucleoli one may suppose that in the root cap cells nucleolar DNA is underreplicated, and to a lesser degree in 5–6 and 7–8 mm segments, while in 3–4 mm segment DNA is overreplicated as compared to meristem cells. Measurements of nucleolar volume, ³H uridine uptake, ³H AMD binding and quantity of granular component, indicate that the most noticeable nucleolar activity takes place in meristematic zone and in root parts showing the highest increase of cytoplasmic volume (3–4 and 5–6 mm segments). ³H leucine is still incorporated intensely into 7–8 mm segment, in which the concentration of ribosomes is low, however they are present in the form of polysomes. Comparison of ³H thymidine uptake into nuclear DNA with ³H AMD binding and ³H uridine incorporation into nuclei indicates that endomitotic DNA replication results in an increase of DNA template activity in root cap cells as well as in 3–4 and 5–6 mm segments; in the 7–8 mm segment binding of ³H AMD slightly decreases, while ³H uridine incorporation is considerably reduced. Divergence between the ploidy state, ³H AMD binding and ³H uridine incorporation can be due to the increment of the condensed chromatin area in differentiated cells. Plastids and mitochondria reach full maturity in 3–4 mm segment. The increasing volume density of ER and diminishing volume density of Golgi structures is accompanied by differentiation of cortex cells.This work was partly supported by Polish Academy of Sciences, Botanical Committee, Grant 217/II     

An ultrastructural study of differentiation of pyriform cells and their contribution to oocyte growth in representative squamata.

An electron microscopic study of the differentiation of pyriform cells and their contribution to oocyte growth in three lizards (Tarentola mauritanica, Cordylus wittifer, Platysaurus intermedius) and one colubrid snake (Coluber viridiflavus) revealed that pyriform cells differentiate from small follicle cells via intermediate cells after establishing an intercellular bridge with the oocyte (see also Hubert: Bull Soc Zool Fr 102:151-158, 1977; Filosa et al: J Embryol Exp Morphol 54:5-15 1979; Klosterman: J Morphol 192:125-144, 1987). Once differentiated, pyriform cells display ultrastructural features indicative of synthetic activity, including abundant ribosomes, Golgi membranes, vacuoles, mitochondria, and lipid droplets. These cellular components extend to the apex of the cell at the level of the intercellular bridge, suggesting that constituents of pyriform cells may be transferred to the oocyte. Furthermore, we demonstrate for the first time that pyriform cells incorporate exogenous yolk. The yolk is segregated inside maturing yolk granules that form in the pyriform cell in the same manner as described for vitellogenic oocytes in non-mammalian vertebrates (see Wallace: Developmental Biology, A Comprehensive Synthesis 127-177, 1985). It is the first clear evidence that pyriform cells and the oocyte may fulfill similar vitellogenic functions. The establishment of an intercellular bridge may represent a crucial event in the development of an integrated system in which pyriform cells and oocyte cooperate.     

Spatial and temporal aspects of cell separation in the foliar abscission zones ofImpatiens sultani Hook

Summary The abscission of leaves fromImpatiens sultani Hook. occurs as the direct result of the weakening of a narrow band of cells running transversely across the base of the petiole. This loss of strength of the abscission zone is due to the breakdown of the central cell wall in two or three layers of cells. The process of wall degeneration is first visible 13 hours after the induction of abscission in a small group of cells found just below the concave groove on the adaxial side of the petiole. As the abscission zone gets progressively weaker the area of cells showing wall breakdown expands, spreading through the parenchyma to the lower side of the stele. The walls of the collenchyma and epidermis along the sides and base of the petiole and the central vascular tissues are the last to break down. Experiments in which the abscission zone was dissected into small pieces were undertaken to investigate whether cell wall hydrolysis was a contagious phenomenon, spreading from cell to cell by direct contact. The results of these investigations indicated that there was little requirement for cell to cell contact in either the temporal or spatial integration of cell wall breakdown.     

Internalization of Sambucus nigra agglutinins I and II in insect midgut CF-203 cells

In this project, the uptake mechanisms and localization of two lectins from Sambucus nigra, further referred to as S. nigra agglutinin (SNA)‐I and SNA‐II, into insect midgut CF‐203 cells were studied. SNA‐I is a chimeric lectin belonging to the class of ribosome‐inactivating proteins, whereas SNA‐II is a hololectin devoid of enzymatic activity. Internalization of the fluorescein isothiocyanate‐labeled lectin was investigated using confocal microscopy. Both lectins were internalized into the cytoplasm of CF‐203 cells at similar rates. Preexposure of the insect midgut cells to specific inhibitors of clathrin‐ and caveolae‐mediated endocytosis resulted in an inhibition of lectin uptake in CF‐203 cells and caspase‐induced cytotoxicity caused by SNA‐I and SNA‐II, confirming the involvement of both endocytosis pathways. Further studies demonstrated that the uptake mechanism(s) for both lectins required phosphoinositide 3‐kinases, but did not depend on the actin cytoskeleton. Since the hololectin SNA‐II apparently uses a similar endocytosis pathway as the chimerolectin SNA‐I, it can be concluded that the endocytosis process mainly relies on the carbohydrate‐binding activity of the lectins under investigation. © 2011 Wiley Periodicals, Inc.     

Nondestructive FTIR monitoring of leaf senescence and elicitin-induced changes in plant leaves

The applicability of the FTIR attenuated total reflectance technique for in situ monitoring of plant physiological processes such as leaf senescence and aging has been examined. Difference spectra obtained by subtracting the spectrum of the young plant leaf from that of the older one revealed positive bands at 1650-1500 cm(-1), indicating a higher relative concentration of phenolics in the older leaves of both black cherry and sweet pepper bush leaves. Prolonged physiological stress of tobacco leaves exhibited a progressive time-dependent increase of the absorbance at around 3475 cm(-1), corresponding to hydroxyl functional groups. Absorption changes were also observed between 1650 and 1500 cm(-1), which are likely to correspond to phenolics. The characteristic changes of the FTIR absorbance spectra resulting from physiological and induced aging were detected also as a response to treatment with a recombinant alpha-elicitin, cinnamomin. This allowed the first quantification of the biological activity of a recombinant elicitin using a spectroscopic method. We suggest that FTIR spectroscopy provides important information about physiological events occurring in plant tissue in vivo, and it could be useful for the in situ characterization of the plant responsiveness to fungal toxins such as elicitins.     

The roles of abscisic acid and ethylene in the abscission and senescence of cocoa flowers

Cocoa flowers have a limited period of longevity; more than 90% of unpollinated flowers abscised within 32 h after anthesis. Abscisic acid (ABA) levels increased significantly prior to abscission. By 21 h after anthesis, ABA levels had increased almost 10-fold, and by 32 h flowers had 20-fold higher levels of ABA than at anthesis. Fluridone completely inhibited both the increase in ABA, the formation of an abscission zone, and the abscission and senescence of flowers. In contrast, ethylene production increased only slightly 21 h after anthesis and was only 2-fold higher after 32 h. Aminoethoxyvinylglycine (AVG) delayed but did not prevent abscission. In cocoa flowers, ABA is the primary regulator of abscission; ethylene accelerates abscission but only in the presence of ABA. Naphthalene acetic acid (NAA) treatment of flowers at anthesis prevented abscission zone formation and flower abscission, but did not induce fruit set. All parts of the NAA-treated flower except the pedicel senesced after 6 days. NAA+AVG treatment only delayed, whereas fluridone treatment completely prevented flower senescence.     

Concentrations of abscisic Acid and indoleacetic Acid in cotton fruits and their abscission zones in relation to fruit retention

An experiment was conducted with field-grown cotton (Gossypium hirsutum L.) to determine the effects of drought and an increase in available photosynthate on the abscisic acid (ABA) and indoleacetic acid (IAA) contents of 3-day-old bolls and their abscission zones. Photosynthate availability was manipulated by removing about two-thirds of the plants to permit increased irradiance, and thus photosynthesis, in the plant canopy. The demand for photosynthate was decreased by removing all bolls from the remaining plants. The thinning and defruiting operations were performed about 3 weeks after first flower. Control plants were neither thinned nor defruited. Effects of water deficit were observed by making three harvests at different times during a 2-week irrigation cycle. Increasing the availability of photosynthate increased boll retention, but had relatively little effect on the concentrations of ABA and IAA in bolls. However, it did increase the concentration of IAA in abscission zones. Water deficit increased the ABA content of bolls and abscission zones and decreased the IAA content of bolls and abscission zones. Across all treatments, the IAA content of abscission zones was positively correlated, and the ABA content of bolls was negatively correlated, with boll retention. The results indicate that stresses change the hormonal balance in ways that are consistent with observed increases in fruit abscission.     

The effects of Sambucus nigra polyphenols on oxidative stress and metabolic disorders in experimental diabetes mellitus

The paper focuses on certain natural polyphenolic extracts from common elder fruit (Sambucus nigra), and also on their effects in diabetes mellitus. The results reveal that the glycosylated hemoglobin values are much higher in the diabetic group and they are significantly lower in the group protected by polyphenols. The natural polyphenol compounds reduce the lipids peroxides, neutralize the lipid peroxil radicals and inhibit the LDL oxidation. Following the perturbation of the lipid metabolism in the diabetic rats, atherogen risk has significantly increased values in comparison to the rats from the witness groups. It is found that due to the polyphenolic protection of the rats from the diabetic group treated with polyphenols, the atherogen risk is preserved at normal limits. The serum activity of glutathione-peroxidase and superoxide-dismutase has significantly lower values in the diabetic group as compared to the group protected by polyphenols. Through the hypoglycemiant, hypolipemiant and antioxidant effects,Sambucus nigra represents a possible dietary adjunct for the treatment of diabetes and a potential source for the discovery of new orally active agent(s) for future diabetes therapy. Understanding the mechanism through which the natural polyphenols have effects on the functionality of the endothelium cells, including on the membrane sensitivity and intracellular signalling, could represent a new way of therapeutically approaching chronic metabolic diseases and cardiovascular illnesses.     

Enzymatic and anatomical changes in abscission zone cells of apple fruits induced by Ethephon

The enhancement of fruit abscission zone formation with ethephon treatment caused an increase in soluble proteins, endo-cellulase, exo-polygalacturonase and peroxidase activities. Exo-cellulase and endo-polygalacturonase did not show any relationship with apple abscission. The separation of cells initiated in the cortex region and progressed towards vascular tissue. Cell separation in the cortex appeared to be due to dissolution of middle lamella but vascular tissues ruptured mechanically.     

Quantitative studies on the ultrastructural differentiation and growth of mammalian cardiac muscle cells. The atria and ventricles of the cat

Ultrastructural differentiation of cardiac muscle cells in the bilateral atria and ventricles of the cat at 1, 16, 25 and 40 days and 6 months after birth was studied by morphometry on electron micrographs. At the newborn stage, no T-tubule was found in the ventricular muscle cells, but specific granules were already noted in the atrial myocytes. The cell diameter of the ventricular myocardium was greater than that of the atrium at this stage. The T-tubule was first recognized in the ventricular muscle cells at day 16, at which stage the area occupied by the mitochondria and glycogen in the atrial muscle cells was definitely found to differ from that in the ventricular muscle cells. Thereafter, the differences in the ultrastructure between the atria and ventricles became more remarkable, particularly in the cell diameter and in the mitochondrial area. The cat cardiac muscle cells are characterized by numerous lipid droplets within the cytoplasm in contrast to those of the rat and the guinea pig.     

Age-related intrinsic changes in human bone-marrow-derived mesenchymal stem cells and their differentiation to osteoblasts

In vivo and in vitro studies indicate that a subpopulation of human marrow-derived stromal cells (MSCs, also known as mesenchymal stem cells) has potential to differentiate into multiple cell types, including osteoblasts. In this study, we tested the hypothesis that there are intrinsic effects of age in human MSCs (17-90 years). We tested the effect of age on senescence-associated beta-galactosidase, proliferation, apoptosis, p53 pathway genes, and osteoblast differentiation in confluent monolayers by alkaline phosphatase activity and osteoblast gene expression analysis. There were fourfold more human bone MSCs (hMSCs) positive for senescence-associated beta-galactosidase in samples from older than younger subjects (P < 0.001; n = 17). Doubling time of hMSCs was 1.7-fold longer in cells from the older than the younger subjects, and was positively correlated with age (P = 0.002; n = 19). Novel age-related changes were identified. With age, more cells were apoptotic (P = 0.016; n = 10). Further, there were age-related increases in expression of p53 and its pathway genes, p21 and BAX. Consistent with other experiments, there was a significant age-related decrease in generation of osteoblasts both in the STRO-1+ cells (P = 0.047; n = 8) and in adherent MSCs (P < 0.001; n = 10). In sum, there is an age-dependent decrease in proliferation and osteoblast differentiation, and an increase in senescence-associated beta-galactosidase-positive cells and apoptosis in hMSCs. Up-regulation of the p53 pathway with age may have a critical role in mediating the reduction in both proliferation and osteoblastogenesis of hMSCs. These findings support the view that there are intrinsic alterations in human MSCs with aging that may contribute to the process of skeletal aging in humans.     

Effects of salts on NADH-malate dehydrogenase activity in celery leaves during their maturation and senescence

The effect of several inorganic salts on the activity of NADH-malate dehydrogenase (MDH) from the petiole and the blade of celery leaves of different age, was investigated. KCl and NaCl, in a concentration range of 100–180 mM, activated the enzyme similarly when given in the assay medium. At higher concentrations, both salts became inhibitory. CaCl₂ and MgCl₂ activated the enzyme at a much lower concentration, with MgCl₂ being the weaker activator. The activation of celery leaf MDH by KCl was found to vary with the leaf age. As the leaf matured, the extracted enzyme exhibited increased activation by KCl. However, the transition from maturity to senescence was accompanied by a decline in the activation of the enzyme by the salt. During senescence of detached leave blades in the dark, the MDH activation by KCl undergoes the same changes found in the intact plant. The changes in MDH activity due to either age or KCl, were a result of changes in the V_max and not of the K_m-value for oxaloacetate. Acceleration of senescence by transient water stress or flooding induced a marked increase in the MDH activation by KCl in vitro.It is suggested that the change in the enzyme response to ions is part of a regulation mechanism by which MDH activity is maintained during various developmental stages of the leaf. However, in accelerated senescence situations, this regulation mechanism is disrupted.     

Phasic changes in immunocytochemical stainability of pituitary luteinizing hormone cells associated with their ultrastructural changes during estrous cycle in the rat

Changes in the immunoreactivity of pituitary luteinizing hormone (LH) cells and in their fine structure were studied in 4-day-cyclic female rats along with the radioimmunoassay of pituitary and serum LH. Pituitary LH increased during diestrus (DE) and in early proestrus (PE) to a maximal level at noon of PE, followed by a marked decrease by 2100 h PE. Serum LH stayed at low levels in estrus (E) and in DE, while they displayed a significant increase at PE. Light microscopic immunocytochemistry distinguished intensely and weakly stained cells using rat LH beta antiserum. The populations of intensely stained cells were 80% at PE, 30% at E and 75% at DE. This suggests that all of the LH cells do not secrete LH synchronously on the afternoon of PE. Immunoreactivity of LH cells was related to the amount of secretory granules stored in the cells as determined by the superimposition technique. Analysis of the LH storage site by the protein A-gold method confirmed that the small secretory granules, which accumulated in LH cells at DE or PE, certainly contain LH. At least two LH cell types were distinguished: one is the oval or polygonal cell with flattened rER numerous mitochondria, abundant small secretory granules (about 200 nm), a well developed Golgi complex, and a round nucleus. The other has similar structural characteristics along with large secretory granules which are more than 300 nm in diameter. At noon of PE almost all of the LH cells were the first type while the second ones were mainly found at DE or E. The relationship of these LH cell types of the male gonadotrophs is discussed.     

The mystique of irreversibility in cyanobacterial heterocyst formation: parallels to differentiation and senescence in eukaryotic cells

The formation of cyanobacterial heterocysts is unique in the prokaryotic world: it is the only irreversible collective process. This terminal differentiation resembles senescence and differentiation in the eukaryotic urkingdom. During their cell cycle eukaryotic cells at the restriction point may reversibly proceed from a vegetative phase (G1) into a quiescent state (G0), and then may irreversibly enter the way towards differentiated or senescent cells. In parallel, at commitment point 1 vegetative cells from filamentous cyanobacteria may reversibly form proheterocysts, and then may proceed irreversibly towards mature heterocysts at commitment point 2. While the signals paving the path for differentiation or senescence in eukaryotes are largely unknown, heterocyst development is clearly triggered by nitrogen starvation. The reasons for the irreversibility in both systems are poorly understood. We discuss these questions, especially in the light of recent advances in the molecular biology of cyanobacteria, with emphasis on self-stabilizing autocatalytic cycles.