Hydrogen fluoride effects on plasma membrane composition and ATPase activity in needles of white pine (Pinus strobus) seedlings pretreated with 12 h photoperiod

 Eastern white pine (Pinus strobus L.) seedlings were pretreated with 12 h photoperiod to induce dormancy. Dormant plants were fumigated with 0.5 ppb (0.4 μg m^–3) or 2.0 ppb (1.6 μg m^–3) hydrogen fluoride (HF) for 2 – 28 days. Plasma membranes were isolated from needles of treated and control seedlings to determine their chemical composition and ATPase activity. For all analyses, only those plants which did not show needle necrosis were selected. The amount of plasma membrane phospholipid expressed on a plasma membrane protein basis was higher after 2 days in the 0.5 ppb HF treatment as compared to controls. After 2 days of 2.0 ppb HF treatment as well as after 8 and 28 days of both HF treatments phospholipid to protein ratios in fluoride treated seedlings were lower as compared to control levels. A decrease in sterol levels could be observed after 2 days in both HF treatments. A large increase in the ratio of sterols to proteins was observed in plasma membranes of eastern white pine seedlings treated with 0.5 ppb HF for 28 days. Increased sterol to phospholipid ratios were observed after 8 and 28 days in 0.5 ppb and after 2 and 8 days of 2.0 ppb HF treatment. A decrease in ATPase activity was observed after 8 days with both fluoride treatments. Drastic increase of ATPase activity was observed after 28 days of HF treated plants. Observed changes of sterol and phospholipid levels after only 2 days of fumigation suggest early fluoride effects on plasma membrane composition during plant dormancy. Received: 25 October 1995 / Accepted: 24 May 1996     

Early effects of triadimefon on water relations, sterol composition and plasma membrane ATPase in white spruce (Picea glauca) seedlings

Seedlings of white spruce ( Picea glauca [Moench] Voss.) were treated with triadimefon solution applied to the soil, and their early responses studied from 12 h to 7 days after treatment. Transpiration rates declined and respiration rates increased immediately after the commencement of triadimefon treatment. Photosynthetic rates declined less than transpiration rates, resulting in an increase in water use efficiency, whereas root and shoot water potentials remained unchanged during the first 5 days of triadimefon treatment. Triadimefon decreased root hydraulic conductivity and inhibited the activity of the plasma membrane ATPase. In addition, triadimefon-treated roots drastically increased the ratios between free sterols and sterol esters and decreased the ratios between sterol esters and acylated sterol glycosides.     

Effects of triadimefon and osmotic stress on plasma membrane composition and ATPase activity in white spruce (Picea glauca) needles

White spruce [ Picea glauca (Moench) Voss.] seedlings were grown in solution culture and treated with 20 mg I^-1 triadimefon [1-(chlorophenoxy)-3,3-dimethyl-1-(1,2,4-triazol-1-yl)-2-butanol] for 4 weeks and then subjected to osmotic stress with polyethylene glycol 3350. Water potentials and electrolyte leakage were measured in control and triadimefon-treated seedlings before and after the plants were subjected to osmotic stress. The plasma membranes were isolated from needles to study their lipid composition and the activity of plasma-membrane bound ATPase. Triadimefon treatment reduced water potentials and increased leakage of electrolytes from seedlings. However, when the seedlings were exposed to osmotic stress, triadimefon-treated plants maintained higher water potentials and leaked less electrolytes compared with the control plants. Both triadimefon and osmotic stress treatments inhibited the activity of plasma membrane-bound ATPase and altered the composition of free sterols in the plasma membranes. Triadimefon-treated plants contained traces of campesterol, which was not present in control. Osmotic stress drastically reduced phospholipid:protein and sterol:protein ratios and increased sterol:phospholipid ratios in the plasma membranes     

Adventitious root initiation in hypocotyl and epicotyl cuttings of eastern white pine (Pinus strobus) seedlings

The present paper reports results of experiments to develop a system for studying adventitious root initiation in cuttings derived from seedlings. Hypocotyl cuttings of 2-week-old eastern white pine (Pinus strobus L.) seedlings were treated for 5 min with 0, 100, 200, 300, 400, 500 or 600 mg l^?1 (0, 0.54, 1.07, 1.61, 2.15, 2.69 or 3.22 mM) 1-naphthaleneacetic acid (NAA) to determine the effect on root initiation. The number of root primordia per cutting was correlated with NAA concentration and the square of NAA concentration. Thus, the number increased from less than one per cutting in the 0 NAA treatment to approximately 40 per cutting at 300 mg l-1 NAA, above which no substantial further increase was observed. The larger number of root primordia formed in response to increasing concentrations of NAA was due to the formation of primordia over a larger proportion of the hypocotyls. Histological analysis of the timing of root primordium formation in hypocotyl cuttings revealed three discernible stages. Progression through these stages was relatively synchronous among NAA-treated hypocotyl cuttings and within a given cutting, but variation was observed in the portion of different cuttings undergoing root formation. Control-treated hypocotyl cuttings formed root primordia at lower frequencies and more slowly than NAA-treated cuttings, with fewer primordia per cutting. Epicotyl cuttings from 11-week-old seedlings also formed adventitious roots, but more slowly than hypocotyl cuttings. NAA treatment of epicotyl cuttings caused more rapid root initiation and also affected the origin of adventitious roots in comparison with nontreated cuttings. NAA-treated epicotyl cuttings formed roots in a manner analogous to that of the hypocotyl cuttings, directly from preformed vascular tissue, while control-treated epicotyl cuttings first formed a wound or callus tissue and subsequently differentiated root primordia within that tissue. This system of inducing adventitious roots in pine stem cuttings lends itself to studying the molecular and biochemical steps that occur during root initiation and development.     

Linkage mapping and genome length in eastern white pine (Pinus strobus L.)

 Haploid linkage analysis of eastern white pine, Pinus strobus L., was carried out using mainly RAPD markers and microsatellite, or simple-sequence-repeat, markers. Ninety one loci mapped to 12 linkage groups of three or more markers. The resulting framework genome map, the first for a soft pine species, contained 69 markers. The map covered 58% of the estimated genome length of 2071 cM(K), with a 95% confidence interval of 1828–2242 cM(K). A systematic comparison of linkage data from eastern white pine, longleaf pine (P. palustris Mill.) and maritime pine (P. pinaster Ait.), gave genome-length estimates for all three species very close to either 2000 cM(K) or 2600 cM(H), depending on whether the Kosambi(K) or Haldane(H) map functions, respectively, were employed. Differences among previous pine genome-length estimates were attributed to the divergent criteria used in the methods of estimation, and indicate the need for the adoption of uniform criteria when performing genome-length estimates. Current data suggest that members of the two pine subgenera, which diverged during the late Mesozoic era, have highly conserved rates of recombination. Received: 5 January 1997/Accepted: 24 January 1997     

Radiation and soil temperature interactions on the growth and physiology of eastern white pine ( Pinus strobus L.) seedlings

A greenhouse experiment was set up during one growing season to test the hypothesis that soil temperature controls a significant part of the light response of eastern white pine (Pinus strobus L.) seedlings that is observed in the field. The experimental design was a three by three factorial split-plot design, with three levels of light availability: 10%, 40% and 80% of full light; and three levels of soil temperature: 16 °C, 21 °C, and 26 °C in the soil at midday. The results show significant interactions between light and soil temperature factors on several variables (gas exchange, root growth, leaf-mass ratio and leaf–mass per unit area), but not on shoot dry mass. These interactions indicate that, in the field, a significant proportion of the light response of young eastern white pine could result from changes in soil temperature, especially under conditions of limiting water availability. Our results suggest that soil temperature must be taken explicitly into account as a driving variable when relating the growth of young eastern white pine to photosynthetic radiation.     

Microsatellite DNA analysis of genetic effects of harvesting in old-growth eastern white pine (Pinus strobus) in Ontario, Canada

Microsatellite DNA markers from 13 simple sequence repeat (SSR) loci were used to compare genetic diversity between preharvest pristine and postharvest residual gene pools of two adjacent virgin, old-growth ( approximately 250 years) stands of eastern white pine (Pinus strobus L.) in Ontario. There was concurrence in genetic diversity changes in the postharvest gene pools of the two stands. The total and mean numbers of alleles detected in each stand were reduced by approximately 26% after tree density reductions of approximately 75%. Approximately 18 and 21% of the low-frequency (0. 25 > P > or = 0.01) alleles and 76 and 92% of the rare (P < 0.01) alleles were lost from residual stands A and B, respectively, after harvesting. Multilocus gametic diversity was reduced by 38 and 85% and genotype additivity by approximately 50% in the residual stands after harvesting. Latent genetic potential of each stand was reduced by approximately 40%. Although heterozygosity was reduced (1-5%) in the postharvest residual stands, the reductions were not substantial and not comparable to those using other genetic diversity measures. The reductions in genetic diversity measures were slightly higher than those theoretically expected in postbottleneck populations according to drift theory. In the absence of substantial gene migration that could ameliorate the genetic losses, the ability of the postharvest white pine gene pools to adapt to changing environmental and disease conditions may have been compromised. The microsatellite DNA results for genetic effects of harvesting in old-growth eastern white pine stands were similar to those that we reported earlier from allozyme analysis (Buchert et al. 1997). The results indicate that silvicultural practices should ensure that the gene pools of remaining pristine old-growth stands are reconstituted in the regenerating stands.     

Cytochemical localization of plasma membrane ATPase activity in plant cells

Summary The cytochemical localization of ATPase activity has been investigated in maize root cells using both lead and cerium-based capture methods. With both methods, staining at the plasma membrane was observed in all cells of the root, although the precipitate obtained with cerium was more uniform and granular than that with lead. Controls using no substrate or no magnesium, -glycerophosphate to replace ATP, vanadate or boiled tissue generally showed little or no staining. However, biochemical studies on purified plasma membrane fractions showed that ATPase activity was markedly inhibited by fixation, particularly by glutaraldehyde, and also by lead and cerium ions. Non-enzymic hydrolysis of ATP by cerium was greater than that by lead. The value and limitations of these procedures for the localization of plasma membrane H⁺-ATPase activity are summarized in relation to previous criticisms of these methods.Abbreviations DTT dithiothreitol - EDTA ethylene diaminetetraacetic acid - GP B-glycerophosphate - PCMBS p-chloromercuribenzene sulphonic acid - PMSF phenylmethylsulphonyl fluoride     

Assessing genetic diversity and structure of fragmented populations of eastern white pine (Pinus strobus) and western white pine (P. monticola) for conservation management

Aims Many pine populations in Canada have fragmented distributions resulting from the effects of glaciations, overharvesting and white pine blister rust infections. Forest fragmentation can modify gene flow and reduce genetic diversity. Selective logging can reduce the density of trees, thereby altering mating patterns and increasing inbreeding. The hypothesis of the present study is that forest fragmentation will not increase inbreeding and will have no effect on genetic diversity parameters in the Canadian Pinus moniticola and P. strobus populations targeted because of (i) the long life span of the pine species, (ii) outbreeding and self-incompatibility of P. monticola and P. strobus and (iii) wind pollination resulting in high gene flow among populations. We studied the genetic diversity of P. strobus across its range in Canada, and we completed a detailed analysis of the genetic structure of P. monticola populations from western Canada using microsatellites genetic markers.Methods Seed samples from 10 P. monticola populations and 10 P. strobus populations were collected from western and eastern Canada, respectively. The mother trees included in seed lots were representative of each stand. Genomic DNA extracted from each sample was amplified with microsatellite primers. The intra- and interpopulation genetic diversity parameters were assessed using Popgene and Genepop softwares and the genetic distances among populations within each species using the PowerMarker software.Important findings Pinus monticola and P. strobus exhibited moderate to high genetic diversity. Also, both species showed low levels of inbreeding despite the geographic isolation and small stand size. Gene flow estimates were high and population differentiation values were relatively low for these fragmented forest sites.     

X-ray microanalytical studies of mineral localization in the needles of white pine (Pinus strobus L.)

Eastern white pine (Pinus strobus L.) shoots from mature trees were collected from two sites of contrasting soil pH: the Glendon campus of York University in Toronto, Ontario (pH 6.7 at 40 cm); and Muskoka near Huntsville, Ontario (pH 4.2 at 40 cm). Needles of ages 1-3 years were removed from the shoots, and the percentage of ash and silica was determined for all ages. Other needles were frozen in liquid nitrogen and kept in a cryo-biological storage system before x-ray microanalysis. Percentages of ash and silica were higher in the needles from Muskoka. Ash and silica increased with needle age for trees from the Muskoka site, but less so at the Toronto site. Of the 13 elements (Na, Mg, Al, Si, P, S, Cl, K, Ca, Mn, Fe, Cu and Zn) detected by microanalysis, Mn, Fe, Cu and Zn were detected in small amounts in the epidermis, endodermis and transfusion tissue (the layer of tracheids and parenchyma immediately surrounding the vascular bundles), and K, P, S and Cl were almost ubiquitous in distribution. Sodium was occasionally detected in the transfusion tissue, and magnesium was concentrated in the endodermal cells. The epidermal walls, transfusion tissue and endodermis were major sites of calcium localization. Silicon was concentrated in the extreme tips of the needles in all tissues, but particularly in the transfusion tissue, and more so in the Muskoka samples. Microanalysis revealed a higher Al content in the Muskoka needles, that Al was concentrated in the needle tips and that the transfusion tissues were major sites of accumulation.     

Stimulation of ATPase activity in barley (Hordeum vulgare) root plasma membrane after treatment of intact tissues and cell free extracts with triacontanol

Ca²⁺- and Mg²⁺-dependent ATPase activity (EC 3.6.1.3) in a plasma membrane-enriched fraction increased rapidly after in vivo application of physiologically active concentrations of triacontanol (TRIA) to the roots of barley ( Hordeum vulgare L. cv. Conquest) seedlings. Ca²⁺- and Mg²⁺-dependent ATPase activity was 64 and 85% higher, respectively, in the roots of seedlings germinated in the presence of growth-promoting concentrations of TRIA compared to controls. The increase in vivo was concentration dependent, with the greatest increase obtained at 2.3 n M TRIA. Maximal stimulation of ATPase activity of excised tissue treated with TRIA coincided with the temperature at which the barley was grown. At this temperature the plasma membrane is primarily in a mixed gel/liquid crystalline state. Pretreatment of barley roots with cyclohexamide did not alter ATPase stimulation by TRIA. Two to three times more [¹⁴C]-TRIA (mg membrane protein)⁻¹ was found associated with plasma membrane-enriched vesicles treated with TRIA than with vesicles enriched for mitochondrial membranes or for vesicles enriched for tonoplast, Golgi and rough endoplasmic reticulum. Both Ca²⁺- and Mg²⁺-dependent ATPase activity increased by 40–60% within 30 min of the addition of 2.3 n M TRIA to cell-free extracts of barley roots. The addition of octacosanol, the C₂₈ analogue of TRIA, to cell-free extracts did not affect metal-dependent ATPase activity. Consistent with many studies in the green-house, simultaneous additions of equimolar amounts of TRIA and octacosanol to cell-free extracts resulted in inhibition of ATPase stimulation by TRIA. TRIA may directly affect plasma membrane function in barley roots.     

Inositol phosphosphingolipid phospholipase C1 regulates plasma membrane ATPase (Pma1) stability in Cryptococcus neoformans

Cryptococcus neoformans is a facultative intracellular pathogen, which can replicate in the acidic environment inside phagolysosomes. Deletion of the enzyme inositol-phosphosphingolipid-phospholipase-C (Isc1) makes C. neoformans hypersensitive to acidic pH likely by inhibiting the function of the proton pump, plasma membrane ATPase (Pma1). In this work, we examined the role of Isc1 on Pma1 transport and oligomerization. Our studies showed that Isc1 deletion did not affect Pma1 synthesis or transport, but significantly inhibited Pma1 oligomerization. Interestingly, Pma1 oligomerization could be restored by supplementing the medium with phytoceramide. These results offer insight into the mechanism of intracellular survival of C. neoformans.     

低温驯化锯缘青蟹鳃抗氧化防护、ATPase及膜脂肪酸组成变化

锯缘青蟹(Scylla serrata)低温环境下生理生化的变化对于理解其低温适应具有重要的意义。本研究中采用生物化学的方法对低温驯化下锯缘青蟹鳃中超氧化物歧化酶(Superoxide dismutase,SOD)、过氧化氢酶(Catalase,CAT)、谷胱甘肽过氧化物酶(Glutathione peroxidase,GPX)的活性,脂质过氧化产物丙二醛(malondialdehyde,MDA)含量,4种ATPase(Na ,K -ATPase,Mg2 -ATPase,Ca2 -ATPase和Ca2 ,Mg2 -ATPase)活性及细胞膜脂肪酸组成进行测定。实验结果显示青蟹鳃中抗氧化酶(SOD、CAT和GPX)活性在3个驯化温度下,随驯化温度的降低而升高。SOD活性在5℃和10℃驯化下显著高于对照组(27℃组)(p<0.01);CAT活性在3个驯化温度下均显著高于对照组(p<0.01或p<0.05);GPX活性仅5℃驯化下显著高于对照组(p<0.01)。MDA含量低温驯化下升高,但仅5℃驯化下显著高于对照组(p<0.01)。鳃中4种ATPase活性均是随驯化温度的降低而升高,并且5℃和10℃驯化下均显著高于对照组(p<0.01或p<0.05)。低温驯化下C18:0、C18:1、C18:2、C18:3、C20:5和C22:6等脂肪酸与对照组相比均发生显著性变化(p<0.01或p<0.05),饱和指数∑SFA/∑UFA显著下降(p<0.01或p<0.05)。低温驯化下锯缘青蟹鳃中抗氧酶及ATPase活性升高,说明其具有明显的温度补偿效应,它是对低温适应的一种积极反应。鳃中MDA低温驯化下积累是活性氧自由基未能被及时清除而产生氧化应激的结果。低温下细胞膜脂肪酸饱和指数降低是维持细胞膜执行正常生理功能的需要。     

The plasma membrane ATPase of Kloeckera apiculata: purification,characterization and effect of ethanol on activity

Partially (6-fold) purified plasma membrane ATPase from an ethanol-sensitive yeast, Kloeckera apiculata, had an optimum pH of 6.0, an optimum temperature of 35°C, a K _m of 3.6 mm ATP and a V _max of 11 mol P_i/min.mg protein. SDS-PAGE of the semi-purified plasma membrane showed a major band of 106 kDa. No in vivo activation of the ATPase by glucose was observed. Although 4% (v/v) ethanol decreased the growth rate by 50% it did not affect the ATPase. Concentrations of ethanol 2% (v/v) did, however, inhibit the enzyme in vitro. The characteristics of the enzyme did not change during growth in the presence of ethanol.     

A link between transport and plasma membrane redox system(s) in carrot cells

Carrot (Daucus carota L.) cells grown in suspension culture oxidized exogeneous NADH. The NADH oxidation was able to stimulate K⁺ (⁸⁶Rb⁺) transport into cells, but it did not affect sucrose transport.N,N'-Dicyclohexyl-carbodiimide, diethylstilbestrol, and oligomycin, which only partially inhibited NADH oxidation, almost completely collapsed the K⁺ (⁸⁶Rb⁺) transport. Vanadate, which is less effective as an ion transport inhibitor, was less effective in inhibiting the NADH-driven transport of K⁺ (⁸⁶Rb⁺).p-Fluormethoxycarbonylcyanide phenylhydrazone inhibits the K⁺ transport over 90% including that induced by NADH. The results are interpreted as evidence that a plasma membrane redox system in root cells is closely associated with the ATPase which can drive K⁺ transport. Because of the inhibitor effects, it appears that membrane components common to the redox system and ATPase function in the transport of K⁺.     

Patterns of growth compensation in eastern white pine (Pinus strobus L.): the influence of herbivory intensity and competitive environments

To investigate complex growth compensation patterns, white pine (Pinus strobus L.) seedlings were clipped to simulate different herbivory levels. Seedlings were growing with different understory competition levels (created through monthly weeding vs no brush control) under a range of overstory canopy closures. Compensation patterns varied for the different growth and size measures. After one growing season, seedlings did not fully compensate for lost biomass regardless of the competitive environments of the seedlings. Although relative height growth was stimulated by light intensity clipping (20-40% of last-year shoots removed), relative diameter growth, total biomass, and biomass growth of seedlings declined sharply with increasing clipping intensity. Likewise, all growth parameters declined with increasing interspecific competition. Results showed that seedlings in highly competitive environments showed smaller growth loss due to clipping than those in competition-free environments, presumably because seedlings experiencing high interspecific competition devoted more energy to maintaining apical dominance and a balanced shoot-root ratio. While competition from canopy trees altered compensatory patterns, competition from understory vegetation only altered the magnitude, but not the patterns, of compensatory growth. We suggest that compensatory growth follows a complex pattern that will vary with the parameters measured, competitive conditions, and clipping intensities. Our results support the assertion that overcompensation may be an adaptation to competitive ability, rather than a response to herbivory itself.     

Calcium signaling and sugar-induced activation of plasma membrane H(+)-ATPase in Saccharomyces cerevisiae cells

In this work, we show that glucose-induced activation of plasma membrane H(+)-ATPase from Saccharomyces cerevisiae is strongly dependent on calcium metabolism and that the glucose sensor Snf3p works in a parallel way with the G protein Gpa2p in the control of the pathway. The role of Snf3p is played by the Snf3p C-terminal tail, since in a strain with the deletion of the SNF3 gene, but also expressing a chimera protein formed by Hxt1p (a glucose transporter) and the Snf3p C-terminal tail, a normal glucose-activation process can be observed. We present evidences indicating that Snf3p would be the sensor for the internal signal (phosphorylated sugars) of this pathway that would connect calcium signaling and activation of the plasma membrane ATPase. We also show that Snf3p could be involved in the control of Pmc1p activity that would regulate the calcium availability in the cytosol.