Different sensitivity to amiloride of body and tail skins of Rana catesbeiana tadpoles during metamorphosis

Regional differences in potential difference and short-circuit current between the body (dorsal) and the tail skin during metamorphosis of Rana catesbeiana tadpoles were investigated. In body skin, the potential difference and the short-circuit current across the skin develop in two successive steps. At stage XX, the potential difference and the short-circuit current across the body skins were amiloride-insensitive (1st step). At stage XXII, however, amiloride-sensitive potential difference and the short circuit current appeared (2nd step). By contrast, in tail skin the potential difference and the short-circuit current remained amiloride-insensitive (1st step) even at stage XXIII. Since the tail regresses after stage XXIII, the appearance of the second step could not be followed in vivo. To determine whether or not the second step can be induced in the tail, tail skin was cultured under conditions where the skin survives for a much longer period than it does in normally developing tadpoles. Such cultured tail skin generated the amiloride-sensitive potential difference and the short-circuit current and cultured body skin also generated them. Therefore, development of the 2nd step in the tail skin may be delayed in vivo. To characterize the differences between body and tail skin, skins were mutally grafted between body and tail at stage XIII–XV. The body skin grafted on the tail underwent both the 1st and 2nd steps by stage XXII, whereas the tail skin grafted on the body only showed the 1st step by the same stage. These results suggest that the regional specificity of the skin is already established before the prometamorphic stage.Abbreviations CMFS Ca²⁺- and Mg²⁺-free saline - CTS charcoal-treated serum - EDTA ethylene diamine tetra-acetate - I current - PD potential difference - R skin resistance - SCC short-circuit current     

Effect of oxytocin on transepithelial transport of water and Na+ in distinct ventral regions of frog skin (Rana catesbeiana)

Thoracic, abdominal, and pelvic fragments of ventral skin of Rana catesbeiana were analysed regarding the effect of oxytocin on: (1) transepithelial water transport; (2) short-circuit current; (3) skin conductance and electrical potential difference; (4) Na⁺ conductance and electrical potential difference; (4) Na⁺ conductance, the electromotive force of Na⁺ transport mechanism, and shunt conductance; (5) short-circuit current responses to fast Na⁺ by K⁺ replacement in the outer compartment, and (6) epithelial microstructure. Unstimulated water and Na⁺ permeabilities were low along the ventral skin. Hydrosmotic and natriferic responses to oxytocin increased from thorax to pelvis. Unstimulated Na⁺ conductance was greater in pelvis than in abdomen, the other electrical parameters being essentially similar in both skin fragments. Contribution of shunt conductance to total skin conductance was higher in abdominal than in pelvic skin. Oxytocin-induced increases of total skin conductance, Na⁺ conductance, and shunt conductance in pelvis were significantly larger than in abdomen. An oscillatory behaviour of the short-circuit current was observed only in oxytocin-treated pelvic skins. Decrease of epithelial thickness and increase of mitochondria-rich cell number were observed from thorax to pelvis. Oxytocin-induced increases of interspaces were more conspicuous in pelvis and abdomen than in thorax.Abbreviations E _Na electromotive force of sodium transport mechansim - G _KCI skin conductance with external KCI Ringer - G _Na sodium conductance (series conductance) - G _shunt shunt pathway conductance - G _total total skin conductance - J _v water flux (in units of volume per area per time) - MRC mitochondria-rich cells - PD potential difference across skin - R _shunt resistance of the shunt pathway - SCC short-circuit current     

Adrenergic receptors and the regulation of vascular resistance in bullfrog tadpoles (Rana catesbeiana)

Summary Vascular adrenergic sensitivity to exogenous catecholamines was examined in tadpoles of the American bullfrog (Rana catesbeiana), ranging from stage III to XIV. Central arterial blood pressure was measured in decerebrate bullfrog tadpoles to determine a reasonable initial infusion pressure. Solutions of epinephrine and phenylephrine were infused into the vasculature of pithed tadpoles, and the resulting changes in vascular resistance (R _v) were used to construct log dose-response relationships. Epinephrine infusion produced a dose-dependent increase in R _v (EC₅₀=5.3·10^-7 M), which could be reversed by sodium nitroprusside (a smooth muscle relaxant) and blocked by phenoxybenzamine (an -adrenergic antagonist). Larval R _v also increased with infusion of the -agonist phenylephrine (EC₅₀=7.4·10⁸ M). Infusion of 10^-6 M isoproterenol (a -agonist) largely reversed the phenylephrine-induced increase in R _v. These results indicate that the capacity exists for both -mediated vasoconstriction and -mediated vasodilation early in bullfrog ontogeny. Neither initial R _v nor the responses to infused epinephrine or phenylephrine were significantly correlated to development over the range of larval stages used in this study.Abbreviations ECG electrocardiogram - EPI epinephrine - ISO isoproterenol - PHE phenylephrine - POB phenoxybenzamine - R _v vascular resistance - SNP sodium nitroprusside     

Ultrastructure of the synaptic ribbons in photoreceptor cells of Rana catesbeiana revealed by freeze-etching and freeze-substitution

Summary The three-dimensional structure of synaptic ribbons in photoreceptor cells of the frog retina was studied with freeze-etching and freeze-substitution methods, combined with a rapid-freezing technique. Although the synaptic ribbon consisted of two electron-dense plaques bisected by an electron-lucent layer in conventional thin sections, such lamellar nature was not so evident in freeze-etched replicas. The cytoplasmic surfaces of the synaptic ribbon presented an extremely regular arrangements of small particles 4–6 nm in diameter. Fine filaments 8–10 nm in diameter and 30–50 nm in length connected synaptic vesicles and the ribbon surface. These connections were mediated by large particles on both ends of the filaments. Approximately 3–5 filaments attached to one synaptic vesicle. Synaptic ribbons were anchored to a characteristic meshwork underlying the presynaptic membrane via another group of similar fine filaments. The meshwork seemed to be an etched replicated image of the presynaptic archiform density observed in thin sections.     

Formation of desmosomes and other contact specializations in cultured skin of the frog (Rana esculenta)

Summary Small trypsinized explants from ventral skin of frogs (Rana esculenta) were maintained in culture for 4 days during which a newly formed epithelium differentiated along the cut edges of the dermis. During the first 6 h adjacent cells produced numerous interdigitating lamellipodia. After 2 days, epithelial polarity was restored by the formation of zonulae occludentes and the epithelial cells were joined by a few small newly formed desmosomes and by numerous interdigitations. Bipartite junctional complexes consisting of a zonula occludens, followed by a series of typical desmosomes, and characteristic of adult frog epidermis were formed only after 4 days. When cultured in the presence of an inhibitor of protein synthesis (cycloheximide) the trypsinized epidermis no longer formed desmosomes. Therefore pools of one or more crucial desmosomal proteins must be very low or non-existent. However, cycloheximide did not prevent the formation of cell contact specializations, consisting of a highly developed system of complex lamellar interdigitations, between adjacent cells.     

Acetylcholinesterase activity in nerve endings of tails of Rana japonica and R. catesbeiana during metamorphosis

Summary In anuran tadpole tails, the myelinated motor nerve fibers branch in the myoseptum to innervate both red and white muscle fibers at, or near, their ends. There are no significant ultrastructural differences between the nerve endings of the two types of muscle fibers.Intense acetylcholinesterase reaction product was observed in synaptic clefts and junctional folds, as well as in transverse tubules. As metamorphosis proceeded, the junctional folds of the nerve endings disappeared, however, acetylcholinesterase reaction product was still observed in the synaptic clefts. As muscle fibers began to degenerate, nerve endings began to separate from them. However, after nerve endings were completely separated from the surfaces, degenerated muscle fibers, synaptic and cored vesicles were still well preserved although no acetylcholinesterase reaction product was found. It seems clear that the mechanism of the muscle degeneration in the tadpole tail during metamorphosis is not the result of the degeneration of its nerve endings.     

Responses of gill mitochondria-rich cells in Mozambique tilapia exposed to acidic environments (pH 4.0) in combination with different salinities

On exposure to hyposmotic acidic water, teleost fish suffer from decreases in blood osmolality and pH, and consequently activate osmoregulatory and acid-base regulatory mechanisms to restore disturbed ion and acid-base balances. In Mozambique tilapia Oreochromis mossambicus exposed to acidic (pH 4.0) or neutral (pH 7.4-7.7) freshwater in combination with 0mM or 50mM NaCl, we examined functional and morphological changes in gill mitochondria-rich (MR) cells. We assessed gene expression of Na(+)/H(+) exchanger-3 (NHE3), Na(+)/Cl(-) cotransporter (NCC), vacuolar-type H(+)-ATPase (V-ATPase) and Na(+)/HCO(3)(-) cotransporter-1 (NBC1) in the gills. The mRNA expression of NHE3 and NCC in tilapia gills were higher in acidic freshwater than in that supplemented with 50mM NaCl, while there was no significant difference in mRNA levels of V-ATPase and NBC1. In addition, immunocytochemical observations showed that apical-NHE3 MR cells were enlarged, and frequently formed multicellular complexes with developed deep apical openings in acidic freshwater with 0mM and 50mM NaCl. These findings suggest that gill MR cells respond to external salinity and pH treatments, by parallel manipulation of osmoregulatory and acid-base regulatory mechanisms.     

Tissue kinetics,ion transport,and recruitment of mitochondria-rich cells in the skin of the toad (Bufo bufo) in response to exposure to distilled water

Mitochondria-rich cells (MRC) of the amphibian epidermis are responsible for active chloride uptake at low external salinity, and new MRCs are recruited in response to exposure to distilled (deionized) water. The time-course of this recruitment, the tissue kinetics and ion transport have been studied in toads (Bufo bufo) immediately before, and after 2,7, and 14 days exposure to distilled water. General epidermal structure was not affected. However, the numbers of MRCs per mm² (D_MRC) increased throughout the experiment as revealed by staining of epidermal sheets with AgNO₃ (Ag) or methylene blue (MB). Part of the increased D_MRC was accounted for by an increase in MRC subpopulation(s) that stained neither with Ag nor MB. The cell birth rate (K_b) decreased and cell loss by moulting (K_d) increased without any significant change in epidermal cell pool size, indicating a reduced apoptotic rate. The increase in D_MRC was accompanied by a 3-fold increase in Cl^- current (I_Cl). At day-2 there was a transient reduction in the I_Cl per MRC. H⁺ secretion was progressively reduced during prolonged exposure to distilled water. Thus, at day-2 MRCs appeared incompletely differentiated as indicated by decreased I_Cl and H⁺ flux per MRC, and by the increased proportion of MRCs unstained by Ag or MB. Full Cl^- (but not H⁺) transport capacity, was restored at day-7. We conclude that increased D_MRC following exposure to low external Cl^-, rather than being due to an increased K_b, is the combined effect of a decreased apoptotic rate and an increased rate of differentiation, where morphological differentiation precedes functional differentiation.Parts of this study have been presented at the 32th International Congress of Physiological Sciences, 1–6 August, 1993, Glasgow, Scotland, and the 19th meeting of the European Study Group for Cell Proliferation, 5–9 October, 1993, Bruges, Belgium     

Calcitonin gene-related peptide and substance P in the pharynx and lung of the bullfrog,Rana catesbeiana

Indirect double immunofluorescence labelling in the pharynx and lung of the bullfrog, Rana catesbeiana, demonstrated the occurrence, distribution, and coexistence of two neuropeptides. In the pharynx, immunoreactive calcitonin gene-related peptide (CGRP) and substance P (SP) were localized in nerve fibers distributed within and just beneath the ciliated epithelium. In the lung, CGRP and SP were localized in nerve fibers in five principal locations: 1) within the smooth muscle layer in the interfaveolar septa; 2) in the luminal thickened edges of the septa; 3) around the pulmonary vasculature; 4) within, and 5) under the ciliated epithelium. Within the smooth muscle layer in the septa, luminal thickened septa, and around blood vessels, almost all fibers showed coexistence of CGRP and SP. Within and just beneath the ciliated epithelium in the thickened septa, all fibers showed coexistence of CGRP and SP. No immunoreactivity for vasoactive intestinal polypeptide, neuropeptide Y, galanin, somatostatin, FMRFamide, and leucine-and methionine-enkephalins was detected in the nerve fibers within the larynx and the lung. Together with our previous data, the present findings suggest that peptidergic mechanisms are involved in the regulation of amphibian respiratory systems throughout their life.     

Prostaglandin E2 enhances the sodium conductance of exocrine glands in isolated frog skin (Rana esculenta)

Summary Prostaglandins are known to stimulate the active transepithelial Na⁺ uptake and the active secretion of Cl^– from the glands of isolated frog skin. In the present work the effect of prostaglandin E₂ (PGE₂) on the glandular Na⁺ conductance was examined. In order to avoid interference from the Na⁺ uptake and the glandular Cl^– secretion the experiments were carried out on skins where the Cl^– secretion was inhibited (the skins were bathed in Cl^– Ringer's solution in the presence of furosemide, or in NO ₃ ^– Ringer's solution), and the active Na⁺ uptake was blocked by the addition of amiloride. Transepithelial current, water flow and ion fluxes were measured. A negative current was passed across the skins (the skins were clamped at –100 mV, basolateral solution was taken as reference). When PGE₂, was added to the skins under these experimental conditions, the current became more negative; this was mainly due to an increase in the Na⁺ efflux. Together with the increase in Na⁺ efflux a significant increase of the water secretion was observed. The water secretion was coupled to the efflux of Na⁺, and when one Na⁺ was pulled from the basolateral to the apical solution via this pathway 230 molecules of water follwed. From the data presented it is suggested that this pathway for Na⁺ is confined to the exocrine glands.     

Ontogeny of substance P-, CGRP-, and VIP-containing nerve fibers in the amphibian carotid labyrinth of the bullfrog,Rana catesbeiana

Summary The ontogeny of substance P, CGRP (calcitonin gene-related peptide), and VIP (vasoactive intestinal polypeptide) containing nerve fibers in the carotid labyrinth of the bullfrog, Rana catesbeiana, was examined by the peroxidase-antiperoxidase method. The time of appearance of these three peptides was different for each. First, CGRP fibers appeared in the wall of the carotid arch and external carotid arteries, and in a thin septum between these two arteries at an early stage of larval development (stage III). At stage V, substance P immunoreactive fibers appeared, and VIP fibers were detected at the early metamorphic stage (stage XXII). Up to the completion of metamorphosis, the number of these fibers remained low. From 1 to 5 weeks after metamorphosis, substance P, CGRP, and VIP fibers increased in number to varying degrees. By 8 weeks after metamorphosis, the distribution and abundance of these fibers closely resembled those of the adults. Some CGRP and VIP immunoreactive glomus cells were found at the stages immediately before and after the completion of metamorphosis. These findings suggest that substance P, CGRP, and VIP fibers during larval development and metamorphosis may be nonfunctional, and start to participate in vascular regulation only after metamorphosis. The transient CGRP and VIP in some glomus cells may be important for the development of the labyrinth, or may take part in vascular regulation through the close apposition of the glomus and smooth muscle cells (g-s connection).     

Immunohistochemistry and nerve lesion experiments on the methionine-enkephalin immunopositive neurons in the small intestine of the bullfrog (Rana catesbeiana)

Summary Nerve elements in the small intestine of the bullfrog. Rana catesbeiana, were studied by immunohistochemistry with anti-methionine enkephalin antisera and by nerve lesion experiments, using laser irradiation. Methionine-enkephalin immunopositive nerve fibers occur in the myenteric plexus, circular muscle layer, submucosa, and mucosa. Immunopositive nerve cell bodies in the myenteric plexus have dendrite-like and a long axon-like processes. In the froglet (3 months after metamorphosis), these axon-like processes lead posteriorly in the nerve strand of the myenteric plexus. Some bifurcate, one branch continuing posteriorly, the other doubling back to lead anteriorly; both form terminal varicose fibers in the circular muscle layer. Nerve lesion experiments, in the adult bullfrog, resulted in accumulations of methionine-enkephalin immunoreactivity at the oral and hinder edges of the laser-irradiated necrotic area; there were sprouting and nonsprouting immunopositive stumps. It is suggested that bidirectional flow of methionine-enkephalin in the myenteric plexus is mediated via the anterior and posterior branches of the axon-like process. The difference in sprouting behavior of immunopositive nerve fiber stumps, after nerve lesion, is discussed with reference to regional differences of the axon-like process.     

Sauvagine-like and corticotropin-releasing factor-like immunoreactivity in the brain of the bullfrog (Rana catesbeiana)

Summary Immunocytochemical methods were used to investigate the occurrence and distribution of sauvagine, corticotropin-releasing factor-, or urotensin I-like immunoreactivities (SVG-ir, CRF-ir, UI-ir, respectively) in the bullfrog (Rana catesbeiana) brain, using specific antisera raised against non-conjugated SVG, ovine CRF, rat/human CRF, and UI. In the hypothalamus, SVG-ir was found in the magnocellular perikarya, in the dorsal and ventral regions of the preoptic nucleus, and in the hypothalamo-hypophyseal projections to the external zone as well as the internal zone of the median eminence, to pars nervosa, and in fibres running from the pars nervosa to the pars intermedia of the pituitary. In contrast, CRF-ir was found only in parvocellular perikarya, mainly localized in the rostro-ventral part of the preoptic nucleus, with fine processes protruding through the ependyma of the third ventricle, fibre projections terminating in the anterior preoptic area and in the neuropil of the periventricular gray, and a caudal projection to the external zone of the median eminence. No CRF-ir staining was seen in the pars nervosa and pars intermedia. The use of UI-specific antisera failed to give a positive response in the frog brain. It is concluded that, in the frog brain, two anatomically different CRF-like (or SVG-like) systems co-exist, comparable to the reported co-existence of UI-ir and CRF-ir neuronal systems in fish brain.     

Regulation of the paracellular Na+ and Cl- conductances by the NaCl-generated osmotic gradient in a manner dependent on the direction of osmotic gradients

In the present study, we investigated the effect of osmolality on the paracellular ion conductance (Gp) composed of the Na⁺ conductance (G_Na) and the Cl⁻ conductance (G_Cl). An osmotic gradient generated by NaCl with relatively apical hypertonicity (NaCl-absorption-direction) induced a large increase in the G_Na associated with a small increase in the G_Cl, whereas an osmotic gradient generated by NaCl with relatively basolateral hypertonicity (NaCl-secretion-direction) induced small increases in the G_Na and the G_Cl. These increases in the Gp caused by NaCl-generated osmotic gradients were diminished by the application of sucrose canceling the NaCl-generated osmotic gradient. The osmotic gradient generated by basolateral application of sucrose without any NaCl gradients had little effects on the Gp. However, this basolateral application of sucrose produced a precondition drastically quickening the time course of the action of the NaCl-generated osmotic gradient on the Gp. Further, we found that application of the basolateral hypotonicity generated by reduction of NaCl concentration shifted the localization of claudin-1 to the apical from the basolateral side. These results indicate that the osmotic gradient regulates the paracellular ion conductive pathway of tight junctions via a mechanism dependent on the direction of NaCl gradients associated with a shift of claudin-1 localization to the apical side in renal A6 epithelial cells.     

Salinity tolerance and structure of external and internal gills in tadpoles of the crab-eating frog,Rana cancrivora

Summary Salinity tolerance and histology of gills were studied in Rana cancrivora larvae. The tadpoles at the external gill stages (W stages 21–22) were able to survive in media containing up to 40% seawater, but died in water of higher salinity. Their external gills appear to have no critical role in adaptation to seawater. However, advanced tadpoles with internal gills (T-K stages I–XVIII) were able to tolerate 50% or higher seawater. In the internal gills, there are numerous mitochondriarich cells (MR cells) scattered on the ventral and lateral epithelia of the gill arches and the gill tufts in both freshwater-and seawater-acclimated tadpoles. In freshwater-acclimated tadpoles there are three types of MR cell: (1) microplicated, (2) microvillous, and (3) apically vacuolated. In tadpoles acclimated to dilute seawater, the ratio of type-1 to type-2 cells is lower, although all three types of MR cell are present. In 60%-seawater-acclimated tadpoles, a few MR cells with a lumen and concave cytoplasm at the apical membrane (type 4) are present. The changes in MR cell morphology under ambient conditions of low or high salinity may reflect alterations in the physiological roles of the gills with regard to transport of ions.     

Identification and structure-activity relationship of an antimicrobial peptide of the palustrin-2 family isolated from the skin of the endangered frog Odorrana ishikawae

Recently, we identified nine novel antimicrobial peptides from the skin of the endangered anuran species, Odorrana ishikawae, to assess its innate immune system. In this study an additional antimicrobial peptide was initially isolated based on antimicrobial activity against Escherichia coli. The new antimicrobial peptide belonging to the palustrin-2 family was named palustrin-2ISb. It consists of 36 amino acid residues including 7 amino acids C-terminal to the cyclic heptapeptide Rana box domain. The peptide's primary structure suggests a close relationship with the Chinese odorous frog, Odorrana grahami. The cloned cDNA encoding the precursor protein contained a signal peptide, an N-terminal acidic spacer domain, a Lys-Arg processing site and the C-terminal precursor antimicrobial peptide. It also contained 3 amino acid residues at the C-terminus not found in the mature peptide. Finally, the antimicrobial activities against four microorganisms (E. coli, Staphylococcus aureus, methicillin-resistant S. aureus and Candida albicans) were investigated using several synthetic peptides. A 29 amino acid truncated form of the peptide, lacking the 7 amino acids C-terminal to the Rana box, possessed greater antimicrobial activities than the native structure.     

Influence of saline stress on root hydraulic conductance and PIP expression in Arabidopsis

Measurements of the root hydraulic conductance (L0) of roots of Arabidopsis thaliana were carried out and the results were compared with the expression of aquaporins present in the plasma membrane of A. thaliana. L0 of plants treated with different NaCl concentrations was progressively reduced as NaCl concentration was increased compared to control plants. Also, L0 of plants treated with 60 mmol/L NaCl for different lengths of time was measured. Variations during the light period were seen, but only for the controls. A good correlation between mRNA expression and L0 was observed in both experiments. Control plants and plants treated with 60 mmol/L NaCl were incubated with Hg and then with DTT. For these plants, L0 and cell-to-cell pathway contributions to root water transport were determined. These results revealed that in control plants most water movement occurs via the cell-to-cell pathway, thus implying aquaporin involvement. But, in NaCl-stressed plants, the Hg-sensitive cell-to-cell pathway could be inhibited already by the effect of NaCl on water channels. Therefore, short periods of NaCl application to Arabidopsis plants are characterised by decreases in the L0 of roots, and are related to down-regulation of the expression of the PIP aquaporins. This finding indicates that the well known effect of salinity on L0 could involve regulation of aquaporin expression.     

Coexistence of substance P,neuropeptide Y,VIP, and CGRP in the nerve fibers of the carotid labyrinth of the bullfrog,Rana catesbeiana: a double-labelling immunofluorescence study in combination with alternate consecutive sections

Double immunohistochemical staining with rhodamine- and fluorescein isothiocyanate (FITC)-conjugated antisera revealed the coexistence of substance P (SP) and neuropeptide Y (NPY), and SP and calcitonin gene-related peptide (CGRP) in most nerve fibers in the intervascular stroma of the carotid labyrinth of the bull-frog, Rana catesbeiana, although there were a few fibers which showed only SP- or NPY-immunoreactivity. Approximately one third of SP-immunoreactive fibers also showed coexistence with vasoactive intestinal polypeptide (VIP)-immunoreactivity, and a few fibers contained VIP without SP. The combination of the double immunofluorescence technique and alternate consecutive sections further demonstrated the possible coexistence of SP, VIP, NPY, and CGRP. This coexistence of four different peptides in the same nerve fibers was proved by the following two evident facts: 1) some SP fibers which demonstrated coexistence with NPY-immunoreactivity were assumed to be continuous with those showing VIP-immunoreactivity, and 2) almost all of the SP fibers showed coexistence with CGRP-immunoreactivity. By this reasoning, nearly one third of SP fibers may demonstrate coexistence with NPY-, VIP-, and CGRP-immunoreactivities. These multiple peptides might be involved in vascular regulatory function, which is a possible function of the amphibian carotid labyrinth.     

The alpha-subunit of glycoprotein hormones exists in the prolactin secretory granules of the bullforg (Rana catesbeiana) pituitary gland

Summary Our recent finding that the number of immunoreactive -subunit cells was invariably greater than the total number of immunoreactive gonadotropin (GTH) and thyrotropin (TSH) cells in the bullfrog (Rana catesbeiana) pituitary gland raises the possibility that the -subunit also exists in pituitary cells other than GTH and TSH cells. The present study demonstrates that there are a considerable number of immunoreactive prolactin (PRL) cells that are also stained with antibody against the -subunit when adjacent sections are immunocytochemically examined. Neither immunoreactive growth hormone nor adrenocorticotropin cells are stained with the antibody against the -subunit. The specificity of the antibody against the -subunit and of that against PRL was demonstrated by preabsorption test, non-competitive binding test, and immunoblot analysis. Double-immunolabeling with gold particles of different sizes for the -subunit and PRL revealed that most of the immunolabeled PRL-secretory granules are also labeled with the -subunit antibody. The gold particles indicating the presence of the -subunit were mostly found in the peripheral zone of the secretory granules.     

Seasonal changes in apparent hydraulic conductance and their implications for water use of European beech (<Emphasis Type="Italic">Fagus sylvatica</Emphasis> L.) and sessile oak [<Emphasis Type="Italic">Quercus petraea</Emphasis> (Matt.) Liebl] in South Europe

The water status of Fagus sylvatica L. and Quercus petraea (Matt) Liebl. was analysed during a cycle of progressive natural drought in southern Europe. Predawn (Ψ_pd) and midday water potential were measured in transpiring (Ψ_leaf) and non-transpiring leaves (Ψ_xyl). Furthermore, photosynthesis (A), stomatal conductance to water vapour (g_s) and sap flow (F_d) were recorded on the same dates. Apparent leaf specific hydraulic conductance in the soil–plant–air continuum (K_h) and whole tree hydraulic conductance (K_hsf) were calculated by using the simple analogy of the Ohm’s law. K_h was estimated at different points in the pathway as the ratio between transpiration (E) in the uppermost canopy leaves at midday and the gradient of water potential in the different compartments of the continuum soil–roots–stem–branches–leaves. There was a progressive decrease in water potential measured on non-transpiring leaves at the base of tree crown in both species (Ψ^l_xyl) from the beginning of the growing season to the end of summer. A similar decrease was shown in shoot water potential (Ψ^u_xyl) at the uppermost canopy. Predawn water potential (Ψ_pd) was high in both species until late July (28 July); afterwards, a significant decrease was registered in F. sylvatica and Q. petraea with minimum values of −0.81±0.03 and −0.75±0.06 MPa, respectively, by 15 September. In both species, leaf specific hydraulic conductance in the overall continuum soil–plant–air (K_h) decreased progressively as water stress increases. Minimum values of K_h and K_hsf were recorded when Ψ_pd was lower. However, Q. petraea showed higher K_h than F. sylvatica for the same Ψ_pd. The decrease in K_h with water stress was mainly linked to its fall from the soil to the lowermost canopy (K_srs). Nevertheless, a significant resistance in the petiole–leaf lamina (K_pl) was also recorded because significant differences in all dates were found on Ψ between transpiring and non-transpiring leaves from the same shoot. The decline in K_h was followed by an increase in stomatal control of daily water losses through the decrease of stomatal conductance to water vapour (g_s) during the day. It promoted a seasonal increase in the stomatal limitation to carbon dioxide uptake for photosynthesis (A). These facts were more relevant in F. sylvatica, which had concurrently a higher decline in water use at the tree level than Q. petraea. The results showed a strong coupling in F. sylvatica and Q. petraea between processes at leaf and tree level. It may be hypothesised a role of specific hydraulic conductance not only in the regulation of water losses by transpiration but also of carbon uptake.