Modification of Plasma Membrane Organization in Tobacco Cells Elicited by Cryptogein

Lipid mixtures within artificial membranes undergo a separation into liquid-disordered and liquid-ordered phases. However, the existence of this segregation into microscopic liquid-ordered phases has been difficult to prove in living cells, and the precise organization of the plasma membrane into such phases has not been elucidated in plant cells. We developed a multispectral confocal microscopy approach to generate ratiometric images of the plasma membrane surface of Bright Yellow 2 tobacco (Nicotiana tabacum) suspension cells labeled with an environment sensitive fluorescent probe. This allowed the in vivo characterization of the global level of order of this membrane, by which we could demonstrate that an increase in its proportion of ordered phases transiently occurred in the early steps of the signaling triggered by cryptogein and flagellin, two elicitors of plant defense reactions. The use of fluorescence recovery after photobleaching revealed an increase in plasma membrane fluidity induced by cryptogein, but not by flagellin. Moreover, we characterized the spatial distribution of liquid-ordered phases on the membrane of living plant cells and monitored their variations induced by cryptogein elicitation. We analyze these results in the context of plant defense signaling, discuss their meaning within the framework of the “membrane raft” hypothesis, and propose a new mechanism of signaling platform formation in response to elicitor treatment.The adaptive capacity of biological membranes is a primary determinant of cell survival in fluctuating conditions. In particular, membrane physical properties are adjusted in the perception of and response to environmental modifications (including temperature, mechanical, and osmotic stresses) in various organisms (Los and Murata, 2004; Vígh et al., 2007; Verstraeten et al., 2010), including plants (Vaultier et al., 2006; Königshofer et al., 2008). Moreover, it has been shown that modifications of plasma membrane (PM) physical properties induced by pharmacological treatments can trigger signaling events in tobacco (Nicotiana tabacum) suspension cells (Bonneau et al., 2010). This reinforces the need to analyze the relationships between membrane organization and signaling in greater detail.Fluidity, a physical property of the PM, is a measure of the rotational and translational motions of molecules within the membrane, and consequently this reflects the level of lipid order in the bilayer. Lipid order is comprised of structure, microviscosity, and membrane phase; the latter feature includes lipid shape, packing, and curvature (Rilfors et al., 1984; van der Meer et al., 1984; Bloom et al., 1991). Lipid self-association induces a physical segregation into lipid bilayers, wherein a liquid-ordered (Lo) phase coexists with a liquid-disordered (Ld) phase (Veatch and Keller, 2005; Gaus et al., 2006; Klymchenko et al., 2009; Heberle et al., 2010). The Lo phase couples a high rotational mobility with a high conformational order in the lipid acyl chain, two physical properties that could be spatially resolved by fluorescence microscopy (Kubiak et al., 2011). Moreover, some observations indicate that Lo size or proportion could be controlled by temperature or cholesterol content (Roche et al., 2008; Orth et al., 2011).This preferential association of some lipids in complex mixtures has resulted in the “membrane raft” hypothesis within the cell biology field. This theory postulates the existence of small (20–200 nm), short-lived, sterol-, and sphingolipid-enriched Lo assemblies within the membrane. An important feature is that these aggregations are believed to coalesce, upon a biological stimulus, into larger structures whose dynamics can regulate many cellular processes (Simons and Ikonen, 1997; Pike, 2006; Lingwood and Simons, 2010; Simons and Gerl, 2010). An increased resistance to solubilization by detergents of Lo versus Ld phases has led researchers to consider that membrane fractions insoluble to nonionic detergents at low temperatures could contain the putative “raft” fractions. One caveat of this theory is that recovered detergent-insoluble membrane fractions (DIMs) only exist after detergent treatment and do not correspond to the native membrane structure (Lichtenberg et al., 2005). Nevertheless, their significant enrichment in sterols, sphingolipids, and specific subsets of proteins, some of which displaying a clustered distribution within the PM (Simons and Gerl, 2010), has encouraged their use as a biochemical counterpart of Lo microdomains existing in biological membranes.Plant DIMs with a lipid content similar to animal DIMs have been isolated from several species, including tobacco cells, and are enriched in proteins involved in signaling and stress responses (Mongrand et al., 2004; Borner et al., 2005; Morel et al., 2006; Lefebvre et al., 2007; Kierszniowska et al., 2009). Moreover, immunoelectron microscopy experiments have revealed that lateral segregation of lipids and proteins occurs at the nanoscale level at the tobacco PM, thus correlating detergent insolubility with membrane domain localization of presumptive raft proteins (Raffaele et al., 2009; Furt et al., 2010; Demir et al., 2013). Together, these data point to the existence of specialized lipid domains in plants. Concomitantly, the presence of sterol-rich Lo membrane domains was observed in vivo at the tip of the growing pollen tube in Picea meyeri, using both filipin and the fluorescent probe 1-[2-hydroxy-3-(N,N-dimethyl-N-hydroxyethyl)ammoniopropyl]-4-[β-[2-(di-n-butylamino)-6-napthyl]vinyl] pyridinium dibromide (di-4-ANEPPDHQ; Liu et al., 2009). This observation argues in favor of a sterol-dependent organization of ordered domains at the plant PM surface. In addition, the combined use of fluorescent lipid analogs and the environmental dye laurdan has revealed different lipid phases that emerge in the PM of Arabidopsis (Arabidopsis thaliana) protoplasts during restoration of the cell wall (Blachutzik et al., 2012). Despite these details, necessary data concerning the presence and in vivo characterization of Lo domains at a micrometer to nanometer scale are still lacking.The importance of a more refined resolution for observing Lo domains was proposed in several recent reviews (Bagatolli, 2006; Duggan et al., 2008; García-Sáez and Schwille, 2010; Owen et al., 2010a; Stöckl and Herrmann, 2010; Klenerman et al., 2011). Although the physical properties of biological membranes have been studied in situ by various techniques, including two-channel ratiometric microscopy (Owen et al., 2010c) and microscopy imaging of partitioning of fluorescent lipids and proteins (Rosetti et al., 2010) or environmentally sensitive probes (Parasassi et al., 1990; Jin et al., 2006), membrane segregation into microscopic Lo- and Ld-like phases has been difficult to observe in living cells. Furthermore, only a few studies have demonstrated that a microscopic phase separation involving an ordered phase similar to the Lo domain of model membranes could occur in biomembranes using PM giant vesicles (Baumgart et al., 2007; Lingwood et al., 2008; Sengupta et al., 2008). A potentially powerful approach for imaging small ordered membrane domains relies on environment-sensitive probes coupled with fluorescence spectroscopy (Gaus et al., 2003, 2006; Oncul et al., 2010). In particular, analysis of the fluorescence of the di-4-ANEPPDHQ probe, which exhibits an emission shift independent of local chemical composition under different lipid packing conditions (Jin et al., 2005; Demchenko et al., 2009; Dinic et al., 2011), recently enabled the imaging of plant membrane domains at the micrometer scale (Liu et al., 2009). The relevance of this approach has been confirmed by mapping membrane domains using generalized anisotropy-based images of di-4-ANEPPDHQ-stained T cell immunological synapses (Owen et al., 2010c), together with the characterization of membrane organization of nonadherent cells (such as living zebrafish embryo tissues) labeled with this dye (Owen et al., 2012a).The function of dynamic PM compartmentalization in the detection and transduction of environmental signals in plant cells has only recently begun to emerge, along with a crucial role for sterols in this organization (for review, see Zappel and Panstruga, 2008; Mongrand et al., 2010; Simon-Plas et al., 2011). These observations make it indispensable to align how the surface membrane of living cells might reorganize during signaling with the membrane raft hypothesis. To investigate possible modifications of membrane organization during the initial steps of plant defense signaling, tobacco cells were treated with two well-described elicitors of defense reaction, cryptogein, a small protein able to trigger an hypersensitive reaction (HR) and an acquired resistance in tobacco plants (Ponchet et al., 1999; Garcia Brugger et al., 2006) together with a widely described signaling cascade in tobacco suspension cells, and flg22 (a 22-amino acid peptide corresponding to a conserved domain of bacterial flagellin). The latter peptide is also a potent elicitor in plants, yet it does not induce an HR type of necrosis (Gomez-Gomez and Boller, 2002; Chinchilla et al., 2007). The study of cryptogein response reveals that the earliest steps of the signal transduction pathway mainly involve PM activities (Ponchet et al., 1999; Garcia-Brugger et al., 2006). How the PM is laterally organized and possibly reorganized in response to this stress so it can efficiently trigger a signaling cascade remains unknown.Here, we have developed a confocal multispectral microscopy approach to generate in vivo ratiometric pictures of large areas of the tobacco cell PM labeled with di-4-ANEPPDHQ, allowing the in vivo characterization of the global level of order of this membrane. Although an increase in the proportion of ordered phase within the membrane transiently occurred in the early steps of the cryptogein and flg22 signaling cascades, the fluorescence recovery after photobleaching (FRAP) technique revealed an increase in PM fluidity induced by cryptogein, but not by flagellin. Moreover, we characterized the spatial distribution of Lo phases on the membrane of living plant cells and monitored the variations induced by cryptogein elicitation. The results are discussed within the framework of the “membrane raft” hypothesis, in which we propose a new mechanism of signaling platform formation in the context of plant defense.     

拟南芥角果衰老过程中膜脂的变化

角果发育对某些物种的生殖发育具有重要的作用。拟南芥种子附着在角果里,角果在早期发育时进行光合作用,角果成熟后开裂散落种子之前,其细胞会经历一个衰老的过程。一般植物细胞在衰老过程中要经历膜脂降解的过程,但是角果细胞衰老过程仍未知。通过比较角果衰老过程中拟南芥野生型(WS)及与膜脂代谢密切相关的磷脂酶Dδ缺失突变体(PLDδ KO)中膜脂分子的组成情况、膜脂含量、相对含量及双键指数值,结果发现,在拟南芥角果衰老过程中：(i)质体膜脂和质体外膜脂显著下降;(ii)不同膜脂降解速率不一样,质体膜脂的降解比质体外膜脂的降解快;(iii)总的双键指数DBI下降;(iv)磷脂酶Dδ缺失突变体(PLDδ KO)的角果膜脂组成的基本水平和变化样式与野生型(WS)非常相似。结果说明,角果在衰老过程中发生了膜脂的激烈降解。据此推测：(i) 膜脂水解产物可能转移到种子中用于储藏脂三酰甘油的合成;(ii) 质体膜脂相对含量下降和质体外膜脂相对含量上升导致了总的DBI下降;(iii) PLDδ参与了角果衰老中的膜脂代谢。     

Alteration of Plasma Membrane Organization by an Anticancer Lysophosphatidylcholine Analogue Induces Intracellular Acidification and Internalization of Plasma Membrane Transporters in Yeast

The lysophosphatidylcholine analogue edelfosine is a potent antitumor lipid that targets cellular membranes. The underlying mechanisms leading to cell death remain controversial, although two cellular membranes have emerged as primary targets of edelfosine, the plasma membrane (PM) and the endoplasmic reticulum. In an effort to identify conditions that enhance or prevent the cytotoxic effect of edelfosine, we have conducted genome-wide surveys of edelfosine sensitivity and resistance in Saccharomyces cerevisiae presented in this work and the accompanying paper (Cuesta-Marbán, Á., Botet, J., Czyz, O., Cacharro, L. M., Gajate, C., Hornillos, V., Delgado, J., Zhang, H., Amat-Guerri, F., Acuña, A. U., McMaster, C. R., Revuelta, J. L., Zaremberg, V., and Mollinedo, F. (January 23, 2013) J. Biol. Chem. 288,), respectively. Our results point to maintenance of pH homeostasis as a major player in modulating susceptibility to edelfosine with the PM proton pump Pma1p playing a main role. We demonstrate that edelfosine alters PM organization and induces intracellular acidification. Significantly, we show that edelfosine selectively reduces lateral segregation of PM proteins like Pma1p and nutrient H⁺-symporters inducing their ubiquitination and internalization. The biology associated to the mode of action of edelfosine we have unveiled includes selective modification of lipid raft integrity altering pH homeostasis, which in turn regulates cell growth.     

Binding of Plasma Membrane Lipids Recruits the Yeast Integral Membrane Protein Ist2 to the Cortical ER

Recruitment of cytosolic proteins to individual membranes is governed by a combination of protein–protein and protein–membrane interactions. Many proteins recognize phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂] at the cytosolic surface of the plasma membrane (PM). Here, we show that a protein–lipid interaction can also serve as a dominant signal for the sorting of integral membrane proteins. Interaction with phosphatidly-inositolphosphates (PIPs) at the PM is involved in the targeting of the polytopic yeast protein Ist2 to PM-associated domains of the cortical endoplasmic reticulum (ER). Moreover, binding of PI(4,5)P₂ at the PM functions as a dominant mechanism that targets other integral membrane proteins to PM-associated domains of the cortical ER. This sorting to a subdomain of the ER abolishes proteasomal degradation and trafficking along the classical secretory (sec) pathway. In combination with the localization of IST2 mRNA to the bud tip and other redundant signals in Ist2, binding of PIPs leads to efficient accumulation of Ist2 at domains of the cortical ER from where the protein may reach the PM independently of the function of the sec-pathway.     

Changes in the Physical State of Membrane Lipids during Senescence of Rose Petals

Changes in the physical state of microsomal membrane lipids during senescence of rose flower petals (Rosa hyb. L. cv Mercedes) were measured by x-ray diffraction analysis. During senescence of cut flowers held at 22°C, lipid in the ordered, gel phase appeared in the otherwise disordered, liquid-crystalline phase lipids of the membranes. This was due to an increase in the phase transition temperature of the lipids. The proportion of gel phase in the membrane lipids of 2-day-old flowers was estimated as about 20% at 22°C. Ethylene may be responsible, at least in part, for the increase in lipid transition temperature during senescence since aminooxyacetic acid and silver thiosulfate inhibited the rise in transition temperature. When flowers were stored at 3°C for 10 to 17 days and then transferrd to 22°C, gel phase lipid appeared in membranes earlier than in freshly cut flowers. This advanced senescence was the result of aging at 3°C, indicated by increases in membrane lipid transition temperature and ethylene production rate during the time at 3°C. It is concluded that changes in the physical state of membrane lipids are an integral part of senescence of rose petals, that they are caused, at least in part, by ethylene action and that they are responsible, at least in part, for the increase in membrane permeability which precedes flower death.     

Adaptive Changes in Membrane Lipids of Barophilic Bacteria in Response to Changes in Growth Pressure

The lipid compositions of barophilic bacterial strains which contained docosahexaenoic acid (DHA [22:6n-3]) were examined, and the adaptive changes of these compositions were analyzed in response to growth pressure. In the facultatively barophilic strain 16C1, phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) were major components which had the same fatty acid chains. However, in PE, monounsaturated fatty acids such as hexadecenoic acid were major components, and DHA accounted for only 3.7% of the total fatty acids, while in PG, DHA accounted for 29.6% of the total fatty acids. In response to an increase in growth pressure in strain 16C1, the amounts of saturated fatty acids in PE were reduced, and these decreases were mainly balanced by an increase in unsaturated fatty acids, including DHA. In PG, the decrease in saturated fatty acids was mainly balanced by an increase in DHA. Similar adaptive changes in fatty acid composition were observed in response to growth pressure in obligately barophilic strain 2D2. Furthermore, these adaptive changes in response were also observed in response to low temperature in strain 16C1. These results confirm that the general shift from saturated to unsaturated fatty acids including DHA is one of the adaptive changes in response to increases in pressure and suggest that DHA may play a role in maintaining the proper fluidity of membrane lipids under high pressure.     

Changes in Membrane Lipids of Roots Associated with Changes in Permeability: I. EFFECTS OF UNDISSOCIATED ORGANIC ACIDS

Previous work has shown that undissociated forms of organic acids, such as formic, acetic, and propionic acids, increase the permeability of barley roots to ions. The work here was undertaken to test whether these undissociated acids affect the lipids from the root membranes in such a way as to account for the permeability increase. Relative amounts of the principal fatty acids from barley root membranes were measured as a function of organic acid concentration, pH, and time of treatment of barley roots under conditions similar to those of the previous studies.     

Organization of G Proteins and Adenylyl Cyclase at the Plasma Membrane

There is mounting evidence for the organization and compartmentation of signaling molecules at the plasma membrane. We find that hormone-sensitive adenylyl cyclase activity is enriched in a subset of regulatory G protein-containing fractions of the plasma membrane. These subfractions resemble, in low buoyant density, structures of the plasma membrane termed caveolae. Immunofluorescence experiments revealed a punctate pattern of G protein α and β subunits, consistent with concentration of these proteins at distinct sites on the plasma membrane. Partial coincidence of localization of G protein α subunits with caveolin (a marker for caveolae) was observed by double immunofluorescence. Results of immunogold electron microscopy suggest that some G protein is associated with invaginated caveolae, but most of the protein resides in irregular structures of the plasma membrane that could not be identified morphologically. Because regulated adenylyl cyclase activity is present in low-density subfractions of plasma membrane from a cell type (S49 lymphoma) that does not express caveolin, this protein is not required for organization of the adenylyl cyclase system. The data suggest that hormone-sensitive adenylyl cyclase systems are localized in a specialized subdomain of the plasma membrane that may optimize the efficiency and fidelity of signal transduction.     

Conformational Changes Produced by ATP Binding to the Plasma Membrane Calcium Pump

The aim of this work was to study the plasma membrane calcium pump (PMCA) reaction cycle by characterizing conformational changes associated with calcium, ATP, and vanadate binding to purified PMCA. This was accomplished by studying the exposure of PMCA to surrounding phospholipids by measuring the incorporation of the photoactivatable phosphatidylcholine analog 1-O-hexadecanoyl-2-O-[9-[[[2-[¹²⁵I]iodo-4-(trifluoromethyl-3H-diazirin-3-yl)benzyl]oxy]carbonyl]nonanoyl]-sn-glycero-3-phosphocholine to the protein. ATP could bind to the different vanadate-bound states of the enzyme either in the presence or in the absence of Ca²⁺ with high apparent affinity. Conformational movements of the ATP binding domain were determined using the fluorescent analog 2′(3′)-O-(2,4,6-trinitrophenyl)adenosine 5′-triphosphate. To assess the conformational behavior of the Ca²⁺ binding domain, we also studied the occlusion of Ca²⁺, both in the presence and in the absence of ATP and with or without vanadate. Results show the existence of occluded species in the presence of vanadate and/or ATP. This allowed the development of a model that describes the transport of Ca²⁺ and its relation with ATP hydrolysis. This is the first approach that uses a conformational study to describe the PMCA P-type ATPase reaction cycle, adding important features to the classical E₁-E₂ model devised using kinetics methodology only.     

Changes in Chloroplast Membrane Lipids during Adaptation of Barley to Extreme Salinity

During adaptation of barley (Hordeum vulgare L.) seedlings to extremely high concentrations of sodium chloride in the root space, the content of galactolipids of chloroplast membranes decreased considerably. Alterations in membrane lipids were due to the high concentration of ions rather than to the increase in the water potential. Sodium chloride was accumulated in the leaf cells and affected lipid-synthesizing enzymes such as galactosyl transferase and acylase which are attached to the chloroplast envelope. The return of salt-adapted barley seedlings to a nutrient solution with low salt concentration resulted in a reversal of the observed changes. It is suggested that the decrease in content of galactolipids in biomembranes is one of the factors causing increased salt resistance in barley plants which are adapted to extreme salinity.     

Effect of NaCl and Polyamines on Plasma Membrane Lipids of Wheat Roots

Caryopses of a salt sensitive wheat cultivar (Triticum aestivum L. cv. Giza 163) were presoaked in 2.5 mM putrescine (Put), 5 mM spermidine (Spd) or 2.5 mM spermine (Spm) for 24 h and then subjected to 150 mM NaCl added to the growth medium for 15 d. Effects of NaCl and polyamines (PAs) on plasma membrane (PM) lipids, phospholipids, fatty acids, and free sterols were determined. NaCl treatment caused a decrease in total phospholipids, increase in saturated fatty acids and altered distribution of sterols and phospholipids. NaCl also induced increase in sterol/phospholipid ratio. PAs treatments (particularly Put and Spd) counterbalanced the NaCl deleterious effects on PM lipids.     

Changes in the Plasma Membrane of Escherichia coli During Magnesium Starvation

The effect of Mg(++) starvation on the structure of the Escherichia coli cell membrane was studied with the freeze-etch technique. Special attention was paid to changes within the plane of the membrane, which in normal exponentially growing cells has a netlike arrangement of particles 2 to 6 nm in diameter. During Mg(++) starvation, a paracrystalline particle pattern appeared on the plasma membrane, and large areas devoid of particles were seen. Although these changes are reproducibly associated with Mg(++) starvation of the bacteria, no decrease in the Mg(++) content of the cell envelope per se was detected, even after 24 hr of Mg(++) deprivation. The structural changes caused by Mg(++) deprivation appeared to involve specific and permanent alterations in membrane development. The absence of other nutrients or divalent cations did not induce similar alterations.     

pH-Induced Proton Permeability Changes of Plasma Membrane Vesicles

In vivo studies with leaf cells of aquatic plant species such as Elodea nuttallii revealed the proton permeability and conductance of the plasma membrane to be strongly pH dependent. The question was posed if similar pH dependent permeability changes also occur in isolated plasma membrane vesicles. Here we report the use of acridine orange to quantify passive proton fluxes. Right-side out vesicles were exposed to pH jumps. From the decay of the applied ΔpH the proton fluxes and proton permeability coefficients (P_H+) were calculated. As in the intact Elodea plasma membrane, the proton permeability of the vesicle membrane is pH sensitive, an effect of internal pH as well as external pH on P_H+ was observed. Under near symmetric conditions, i.e., zero electrical potential and zero ΔpH, P_H+ increased from 65 × 10⁻⁸ at pH 8.5 to 10⁻¹ m/sec at pH 11 and the conductance from 13 × 10⁻⁶ to 30 × 10⁻⁴ S/m². At a constant pH _i of 8 and a pH _o going from 8.5 to 11, P_H+ increased more than tenfold from 2 to 26 × 10⁻⁶ m/sec. The calculated values of P_H+ were several orders of magnitude lower than those obtained from studies on intact leaves. Apparently, in plasma membrane purified vesicles the transport system responsible for the observed high proton permeability in vivo is either (partly) inactive or lost during the procedure of vesicle preparation. The residue proton permeability is in agreement with values found for liposome or planar lipid bilayer membranes, suggesting that it reflects an intrinsic permeability of the phospholipid bilayer to protons. Possible implications of these findings for transport studies on similar vesicle systems are discussed. Received: 5 April 1995/Revised: 28 March 1996     

Intake of Xylooligosaccharides Alters the Structural Organization of Liver Plasma Membrane Bilayer

Xylooligosaccharides (XOS) are non-digestible carbohydrate prebiotics that beneficially affect the host by selective stimulation of specific bacteria in the gastro-intestinal tract. The impact of XOS on gastrointestinal microflora and blood lipids is well known but the exact mechanism of action on liver membranes is still unclear. The organization of membrane lipids in domains is known to be important for the proper functioning of various receptors and mechanisms triggering cell signaling. In this study the influence of XOS-enriched diet on the lipid bilayer structure of rat liver plasma membrane was investigated. XOS intake caused a slight decrease of the fluidity of lipid extracts from liver plasma membranes compared to the controls. This observation was based on the increased generalized polarization (GP) and blue shifted emission spectra of Laurdan. The elevated amount of membrane sphingomyelin may be one possible reason for the reported effects. The micron-scale phase separation of the lipid extracts was also investigated by fluorescence microscopy. A different temperature of phase separation and domain pattern was observed in plasma membrane lipid extracts from XOS-fed animals. We presume that it could be assigned to the altered lipid composition of the membrane bilayer, in particular to the changes in the sphingomyelin/cholesterol ratio. All observed alterations are discussed in the light of the impact of XOS on human health and physiology.     

Fusicoccin Binding to its Plasma Membrane Receptor and the Activation of the Plasma Membrane H+-ATPase. II. Stimulation of the H+-ATPase in a Plasma Membrane Fraction Purified by Phase-partitioning

The effect of fusicoccin (FC) on the activity of the PM H⁺-ATPase was investigated in a plasma membrane (PM) fraction from radish seedlings purified by the phase-partitioning procedure. FC stimulated the PM H⁺-ATPase activity by up to 100 %; the effect was essentially on V_max with only a slight decrease of the apparent K_M of the enzyme for ATP. FC-induced stimulation of the PM H⁺-ATPase was evident within the first minute and maximal within five minutes of membrane treatment with the toxin indicating that transmission of the signal from the activated receptor to the PM H⁺-ATPase is very rapid. Both FC-induced stimulation of the PM H⁺-ATPase and FC binding to its receptor decreased dramatically upon incubation of the membranes in ATPase assay medium at 33 °C in the absence of FC, due to the lability of the free FC receptor. FC-induced stimulation of the PM H⁺-ATPase was strongly pH dependent: absolute increase of activity was maximal at pH 7, while percent stimulation increased with the increase of pH up to pH 7.5; FC binding was scarcely influenced by pH in the pH range investigated. Taken as a whole, these results indicate that FC binding is a condition necessary, but not sufficient, for FC-induced stimulation of the PM H⁺-ATPase.     

Deuterium Magnetic Resonance Studies of Senescence-Related Changes in the Physical Properties of Rose Petal Membrane Lipids

The physical properties of membrane lipids in senescing rose (Rosa hybrida L., cv Mercedes) petals were studied by deuterium nuclear magnetic resonance (2H-NMR) and fluorescence depolarization. All of the 2H-NMR spectra arising from deuterated dimyristoylphosphatidylcholine mixed with whole-lipid extracts from membranes of petals of different ages had a shape that is characteristic of liquid-crystalline lipid at 30[deg]C. Arrhenius plots of the moments of the 2H spectra and fluorescence depolarization values measured from 1,6-diphenyl hexatriene-labeled rose petal membrane lipid samples indicated that membrane lipid order increased with decreasing temperature as well as with increasing age of the petals. The latter trend is explained by previously observed increases in fatty acid saturation and increases in the sterol-to-phospholipid ratio that occur in rose petals during senescence. The 2H-NMR spectra obtained at 0[deg]C also contained quadrupolar splitting lines from lipid in the gel phase, confirming the occurrence of this phase in membranes from this tissue.