Movement of [14C]sucrose along the stolon of Saxifraga sarmentosa

Summary The characteristics of ¹³⁷Cs transport along the stolon of Saxifraga previously reported have been confirmed for applied sucrose and natural assimilate. Long-distance transport is strictly unidirectional, with a symmetrical short-distance spread from the point of application. Only the latter takes place in a long piece of excised stolon. Transport is readily reversed when the parent plant is darkened and the daugther, plantlet allowed to photosynthesise. These findings strongly support a mass-flow mechanism for the stolon. They also confirm the value of ¹³⁷Cs as a tracer for assimilate movement, though in contrast to assimilate it suffers appreciable lateral leakage. Pulse labelling of the subtending leaf failed to produce a sharp peak of activity in the stolon. A flattening with time of the ¹⁴C profile is considered to be due to differing linear velocities in parallel sieve tubes.This work formed part of that submitted for the degree, of Ph. D. of the University of London.     

The influence of dinitrophenol on phloem transport along the stolon of Saxifraga sarmentosa

Summary Dinitrophenol in concentrations of 5×10^-3 M applied to the centre 30 cm of 60–70 cm stolons of Saxifraga produces a strong and reversible inhibition of the phloem transport of ¹³⁷Cs or ¹⁴C-assimilates. There is every reason to believe that this effect is localised in the sieve tubes; callose formation does not occur. This evidence is very difficult to reconcile with the Münch hypothesis; it seems on the contrary to demand a theory of active pumping.This work formed part of that submitted for the degree of Ph.D. of London University.     

The effect of nitrogen on the movement of tracers down the stolon of Saxifraga sarmentosa,with some observations on the influence of light

Summary The movement of applied ¹³⁷Cs and of naturally-assimilated ¹⁴C down the long stolon of Saxifraga is strongly inhibited by confining a length of 10 to 30 cm of the stolon in an atmosphere of nitrogen. The inhibition is reversible, normal transport being restored after less then 4 h when the stolon is returned to air from 5 h in nitrogen. Callose formation does not seem to be involved. There is evidence that local darkness has a similar adverse effect on phloem transport.These findings are considered antagonistic to the pressure-flow hypothesis, but favourable to the active mass-flow theories.This work formed part of that submitted for the degree of Ph.D. of the University of London.     

Cyanide Inhibition of Phloem Transport along the Stolon of Saxifraga sarmentosa L

Long thin stolons of Saxifraga were treated with cyanide inboth the solution and gaseous forms; the latter was much moreeffective. Cyanide strongly inhibited the transport of ¹³⁷Csand of natural ¹⁴C-assimilates. As judged by a variety of approaches,including the use of ¹⁴C-cyanide, the inhibition was certainlyeffective in the sieve tubes themselves. Callose formation didnot seem to be promoted. The inhibition was completely reversible,and was never accompanied by a build-up of tracer in, or before,the treated zone; failure to traverse it was not due thereforeto membrane damage and consequent leakage. For these reasonsthe results are held to favour a mechanism of mass flow invokingactive pumping in the sieve tubes.     

The simultaneous movement of two ions in the phloem of the Saxifraga stolon

Summary Paris of tracers were applied simultaneously to the long thin stolons of Saxifraga sarmentosa. After several hours of translocation the very precise pattern of exponential fall-off was examined and interpreted in the light of a model of mass flow with leakage. ⁴²K appears to leak faster than ²²Na; ⁸⁶Rb is very close to ⁴²K. The anion ⁸²Br shows a lower fall-off than ¹³⁷Cs; this is tentatively regarded as due to a much-reduced leakage, though it might imply a higher velocity. The implications of these findings for sieve-tube mechanism are uncertain.This work formed part of that submitted for the degree of Ph.D. of the University of London.     

The effect of low temperatures on the phloem transport of radioactive assimilates in the stolon of Saxifraga sarmentosa L.

Abstract. The movement of naturally assimilated ¹⁴C down the stolon of Saxifraga sarmentosa under the influence of localized cold treatments (0°-10°C) applied over lengths up to 10 cm was followed by finding the distribution of tracer at the termination of the experiment. Inhibition is slight at 10°C and very considerable at 0°C. It is very dependent on the length treated, but is definite even when this is only 0.8 cm. Cooling the daughter plantlet to 0°C has a smaller inhibitory effect than might be expected. This is difficult to understand if 'unloading' is metabolic; perhaps the concentration free energy of the sucrose or other solute can be utilized in the sink terminals on the lines of the chemi-osmotic hypothesis. If so the same source of non-respiratory free energy might be available to energize an active sieve-tube mechanism in mid-path chilling experiments.     

Development of the Sieve Plate in Saxifraga sarmentosa L.

The differentiating sieve plate in the phloem of the stolonof Saxifraga sarmentosa L. was studied with the electron microscope.Development of the pore site begins with differentiation ofa pair of collar-like areas around the plasmodesma which canbe seen in the youngest identifiable sieve plates. Further growthof the collars occurs by deposition of an amorphous substance,presumably caflose. Although the growth of the collars is simultaneouswith the growth of the surrounding cell wall it is rapid atfirst and the pore sites appear asdome-shaped protuberances.It also involves deposition of callose over an increasinglywider area of the cell wall and since the thickening of thenormal cell wall continues only where notcovered by callose,the collars assume a conical form. There seems to be no displacementor lysis of normal cell wall material during growth of the collars.Eventually the growth of the cell wall in thickness overtakesthe pore sites so that when the growth of the cell wall is completethe pore sites appear as depressions in the sieve plate. Theperforation of a pore site is accomplished by widening of theplasmodesmatal cylinder which begins at the middle lamella byremoval of callose. Endoplasmic reticulum is found in closeproximity to the plasmodesma andis believed to penetrate it.     

Translocation in the Stolon of Saxifraga sarmentosa L. The Ultrastructural Background

The ultrastructure of the mature sieve elements of the Saxifragastolon is described. Theseare narrow (3–5 µm) andfairly long (100-300 µm). The sieve plate pores were invariablyfound to be closely-occluded with P-protein. It is argued thatthe double-cutting technique used for excision invalidates thecontention that this must be interpreted as an artifact. TheP-protein filaments appear to consist of a double helix. Stereomicrographs at normal voltages, and at 1 MV in the AEI-EM₇ microscopeare presented.     

The Effect of Inhibitors on Callose Formation in the Sieve Tubes of Saxifraga sarmentosa L.

Electron-microscope studies on stolons undergoing transportinhibition under nitrogen, HCN, and DNP treatment show no evidenceof callose-blockage of the sieve plates. Inhibition must thereforebe interpreted in terms of interruption of the sieve-tube energysupply; this weights the evidence in favour of theories of activemass flow such as those invoking electro-osmosis or proteincontractility.     

Unidirectional movement of an actin filament taking advantage of temperature gradients

An actin filament with heat acceptors attached to its Cys374 residue in each actin monomer could move unidirectionally even under heat pulsation alone, while in the total absence of both ATP and myosin. The prime driver for the movement was temperature gradients operating between locally heated portions on an actin filament and its cooler surroundings. In this report, we investigated how the mitigation of the temperature gradients induces a unidirectional movement of an actin filament. We then observed the transversal fluctuations of the filament in response to heat pulsation and their transition into longitudinally unidirectional movement. The transition was significantly accelerated when Cys374 and Lys336 were simultaneously excited within an actin monomer. These results suggest that the mitigation of the temperature gradients within each actin monomer first went through the energy transformation to transversal fluctuations of the filament, and then followed by the transformation further down to longitudinal movements of the filament. The faster mitigation of temperature gradients within actin monomer helps build up the transition from the transversal to longitudinal movements of the filament by coordinating the interaction between the neighboring monomers.     

Unidirectional flux of phenylalanine into Vero cells. Measurement using paired tracers in perfused cultures

The uptake of phenylalanine by Vero cells in perfused culture was measured using a double-label technique. Cells were anchored in microcarrier beads and maintained in a column perfused at a constant rate. The extracellular tracer [14C]mannitol and the test tracer [3H]phenylalanine were injected as a bolus, and the column effluent was sampled at 10-s intervals. The proportion of the test tracer retained by the cells was calculated by analysis of time-dilution curves of test and reference tracers. Uptake measurements were specific and highly reproducible. Uptake of [3H]phenylalanine was inhibited by unlabelled phenylalanine and by other amino acids that utilize transport system L. This new approach proved useful for rapid measurement of unidirectional uptake, and for determination of kinetics parameters of uptake under steady state conditions. This rapid technique obviates some of the limitations associated with uptake measurements in whole organs and with measurements in conventional cell cultures.     

Genetic Diversity and Spatial Genetic Structure of the Grassland Perennial Saxifraga granulata along Two River Systems

Due to changes in land use, the natural habitats of an increasing number of plant species have become more and more fragmented. In landscapes that consist of patches of suitable habitat, the frequency and extent of long-distance seed dispersal can be expected to be an important factor determining local genetic diversity and regional population structure of the remaining populations. In plant species that are restricted to riparian habitats, rivers can be expected to have a strong impact on the dynamics and spatial genetic structure of populations as they may enable long-distance seed dispersal and thus maintain gene flow between fragmented populations. In this study, we used polymorphic microsatellite markers to investigate the genetic diversity and the spatial genetic structure of 28 populations of Saxifraga granulata along two rivers in central Belgium. We hypothesized that rivers might be essential for gene flow among increasingly isolated populations of this species. Genetic diversity was high (H_S = 0.68), which to a certain extent can be explained by the octoploid nature of S. granulata in the study area. Populations along the Dijle and Demer rivers were also highly differentiated (G”_ST = 0.269 and 0.164 and D_EST = 0.190 and 0.124, respectively) and showed significant isolation-by-distance, indicating moderate levels of gene flow primarily between populations that are geographically close to each other. Along the river Demer population genetic diversity was higher upstream than downstream, suggesting that seed dispersal via the water was not the primary mode of dispersal. Overall, these results indicate that despite increasing fragmentation populations along both rivers were highly genetically diverse. The high ploidy level and longevity of S. granulata have most likely buffered negative effects of fragmentation on genetic diversity and the spatial genetic structure of populations in riparian grasslands.     

Nucleocytoplasmic movement of fluorescent tracers microinjected into living salivary gland cells

The permeability of the nuclear envelop of a somatic cell, the C. thummi larval salivary gland cell, was studied by intracellular microinjection of fluorescent molecular tracers. As shown previously in oocytes (4,5,15,16), the envelop is permeable to a wide variety of materials, including molecules which are large enough to possess condiderable biological specificities and to play important roles in regulation of cellular activities. The envelop exhibits transport selectivity on the basis of size in the range of naturally occurring intracellular materials and it may thus perform important controlling functions in nucleocytoplasmic exchange. The nucleus to cytoplasm movement of in vivo ribonucleoprotein particulates in these synthetically active cells probably requires conformational changes in the particulates and/or the envelope pore complexes; morphological evidence exists for such processess in these cells (20).     

Unidirectional sliding of myosin filaments along the bundle of F-actin filaments spontaneously formed during superprecipitation

I reported previously (Higashi-Fujime, S., 1982, Cold Spring Harbor Symp. Quant. Biol., 46:69-75) that active movements of fibrils composed of F-actin and myosin filaments occurred after superprecipitation in the presence of ATP at low ionic strengths. When the concentration of MgCl2 in the medium used in the above experiment was raised to 20-26 mM, bundles of F-actin filaments, in addition to large precipitates, were formed spontaneously both during and after superprecipitation. Along these bundles, many myosin filaments were observed to slide unidirectionally and successively through the bundle, from one end to the other. The sliding of myosin filaments continued for approximately 1 h at room temperature at a mean rate of 6.0 micron/s, as long as ATP remained in the medium. By electron microscopy, it was found that most F-actin filaments decorated with heavy meromyosin pointed to the same direction in the bundle. Myosin filaments moved actively not only along the F-actin bundle but also in the medium. Such movement probably occurred along F-actin filaments that did not form the bundle but were dispersed in the medium, although dispersed F-actin filaments were not visible under the microscope. In this case, myosin filament could have moved in a reverse direction, changing from one F-actin filament to the other. These results suggested that the direction of movement of myosin filament, which has a bipolar structure and the potentiality to move in both directions, was determined by the polarity of F-actin filament in action.     

Mathematical model of movement of asymmetrical erythrocyte along the capillary

The proposed 3-dimensional mathematical model describes the motion of asymmetrical erythrocytes through capillaries of different sections. The lubrication theory is used to describe the flow of the suspending fluid in the gaps between the cell and the vessel wall. It is shown that the cell velocity and diameter of the capillary defines the value of the resistance of erythrocyte motion.     

Divergent selection along climatic gradients in a rare central European endemic species,Saxifraga sponhemica

Background and Aims The effects of habitat fragmentation on quantitative genetic variation in plant populations are still poorly known. Saxifraga sponhemica is a rare endemic of Central Europe with a disjunct distribution, and a stable and specialized habitat of treeless screes and cliffs. This study therefore used S. sponhemica as a model species to compare quantitative and molecular variation in order to explore (1) the relative importance of drift and selection in shaping the distribution of quantitative genetic variation along climatic gradients; (2) the relationship between plant fitness, quantitative genetic variation, molecular genetic variation and population size; and (3) the relationship between the differentiation of a trait among populations and its evolvability.Methods Genetic variation within and among 22 populations from the whole distribution area of S. sponhemica was studied using RAPD (random amplified polymorphic DNA) markers, and climatic variables were obtained for each site. Seeds were collected from each population and germinated, and seedlings were transplanted into a common garden for determination of variation in plant traits.Key Results In contrast to previous results from rare plant species, strong evidence was found for divergent selection. Most population trait means of S. sponhemica were significantly related to climate gradients, indicating adaptation. Quantitative genetic differentiation increased with geographical distance, even when neutral molecular divergence was controlled for, and Q_ST exceeded F_ST for some traits. The evolvability of traits was negatively correlated with the degree of differentiation among populations (Q_ST), i.e. traits under strong selection showed little genetic variation within populations. The evolutionary potential of a population was not related to its size, the performance of the population or its neutral genetic diversity. However, performance in the common garden was lower for plants from populations with reduced molecular genetic variation, suggesting inbreeding depression due to genetic erosion.Conclusions The findings suggest that studies of molecular and quantitative genetic variation may provide complementary insights important for the conservation of rare species. The strong differentiation of quantitative traits among populations shows that selection can be an important force for structuring variation in evolutionarily important traits even for rare endemic species restricted to very specific habitats.     

Unidirectional peristaltic movement in multisite drug binding pockets of AcrB from molecular dynamics simulations

Tripartite complex AcrB-ToIC, the major efflux system in Escherichia coli, is the principal multidrug transporter in Gram-negative bacteria, which is important in antibiotic drug tolerance. AcrB is a homotrimer that acts as a tripartite complex with the outer membrane channel ToIC and the membrane fusion protein AcrA. Recently, the crystal structures of AcrB bound to the high-molecular-mass drugs rifampicin and erythromycin were reported. Here we performed 20 ns molecular dynamics (MD) simulations of the AcrB-rifampicin-minocycline complex in a lipid bilayer and explicit water. We found that the bound drugs, rifampicin and erythromycin, made a unidirectional peristaltic movement towards the extrusion funnel of ToIC, which was facilitated by the water efflux in the channel of AcrB. With a shift of the Phe-617 loop, rifampicin in the access monomer moved towards the entrance of the distal binding pocket. Minocycline in the binding monomer moved from the distal binding pocket towards the gate of the central funnel. The channel between the entrance and the gate made a concerted opening during the MD simulations, which was helpful for the peristaltic movement. Our results showed that the mutations of Gly616Pro and Gly619Pro prevented the movement of the Phe-617 loop, which indicated the critical role of the flexibility of the Phe-617 loop. In addition, three putative proton translocation channels were proposed based on our results. Our study provided dynamical information and important residues for the peristaltic movement in AcrB, which were critical for substrate uptake and extrusion function.     

Reproductive differentiation in a Saxifraga hirculus population along an environmental gradient on a central Swedish mire

The perennial herbaceous mire plant Saxifraga hirculus was studied in different population densities along an environmental gradient on a mire in central Sweden. Tests were made whether the habitat conditions influenced the balance between vegetative growth of runners and seed production. Along a 30 m long transect the gradient was analysed for vegetation, hydrology, temperature and contents of ions in groundwater and surface peat. Three main zones were distinguished, a spring area, a rich fen area and an intermediate fen area. Range in concentration of ions in groundwater along the transect (mg 1⁻¹): Ca 4–59, Mg 0.3–3.5, K 0.1–3.0, Na 1.0–4.1 and Fe 0.4–4.5. Range in pH: 5.8–7.5. Despite the gradient in ion concentration, the element content in S. hirculus was constant along the transect. Population density reached maximum in the rich fen, 52 floral shoots 0.25 m⁻². In the spring area, S. hirculus was characterized by few flowers per floral shoot, low height, numerous runners and a large total length of runners. In the rich fen, high seed production was characteristic; in the intermediate fen, production of both runners and seeds were low. The flowering of S. hirculus starts early in the spring area. Consequences of this with regard to genetic variation along the transect are briefly discussed.     

Typification of the Linnean species of Saxifraga

WEBB, D. A., 1987. Typification of the Linnean species of Saxifraga. Types (mainly lectotypes) arc assigned to all the species of Saxifraga described by Linnaeus. There are 38 names in all, of which one is now assigned to Bergenia, one is known to be a hybrid, three have been rejected in the past as nomina ambigua, one was later reduced to synonymy by Linnaeus himself, while 32 are accepted as valid species today. Three have been previously typified by Pugsley, one by Steam and one by the author; for the others the types are newly proposed here. Nineteen names are typified from the Linnean herbarium in London (LINN), three from the Linnean herbarium in Stockholm (S), seven from the Burser herbarium at Uppsala (UPS), one from the Tournefort herbarium in Paris (P), and eight from illustrations cited by Linnaeus in synonymy. Lists are appended of all specimens of the genus in the Linnean herbaria in London and Stockholm and the Burser herbarium at Uppsala, and a determination is given for each specimen, though in some cases these can only be tentative. The specimens include 15 species which Linnaeus did not describe, though in the case of five he probably regarded them as varieties.     

Genetic enrichment of the arctic clonal plant Saxifraga cernua at its southern periphery via the alpine sexual Saxifraga sibirica

Isolation of populations at the margins of a species range may lead to decreasing genetic diversity via genetic drift and inbreeding. Hybridization between peripheral populations of two species can, however, counteract genetic impoverishment. The mainly clonal, polyploid plant Saxifraga cernua has a wide arctic distribution but also extends southwards into alpine sites. In the Ural Mountains, its peripheral distribution overlaps with that of its sexually reproducing, diploid relative Saxifraga sibirica, and fertile polyploids of more or less intermediate appearance are found in this overlap zone. We used amplified fragment length polymorphism (AFLP) analysis to address the potential impact of interspecific gene flow on genetic diversity in the peripheral populations. A total of 149 plants from 17 populations along a 1650 km south-north gradient were analysed for 253 markers. The results suggest that three Middle Ural populations containing fertile and morphologically more or less intermediate plants have been affected by hybridization. All of these plants formed a strongly supported (100%) group with S. cernua in a neighbour-joining tree, but their AFLP phenotypes assigned either to S. cernua or to artificial (simulated) F(1) hybrids between S. cernua and S. sibirica in multilocus assignment tests. The three populations were highly diverse with virtually every plant representing a distinct AFLP phenotype, providing additional evidence for formation of later-generation hybrids and/or backcrossing to S. cernua. In contrast, other peripheral populations of S. cernua were typically monoclonal, suggesting that hybridization with S. sibirica can increase genetic diversity in S. cernua at its southern periphery.