Electric and Structural Studies of Hormone Interaction with Chloroplast Envelope Membranes Isolated from Vegetative and Generative Rape

ARTICLE IN PRESS

Journal of Plant Physiology 164 (2007) 861—867

www.elsevier.de/jplph

Electric and structural studies of hormone interaction with chloroplast envelope membranes isolated from vegetative and generative rape

Maria Fileka,Ã, Maria Zembalab, Anna Dudeka, Peter Laggnerc, Manfred Kriechbaumc aInstitute of Plant Physiology, Polish Academy of Sciences, Podłuz˙na 3, 30-239 Krakow, Poland bInstitute of Catalysis and Surface Chemistry Polish Academy of Sciences, Niezapominajek 8, 30-239 Krako´w, Poland cInstitute of Biophysics and X-ray Structure Research, Austrian Academy of Sciences, Schmiedellstrasse 6, 8042 Graz, Austria

Received 7 December 2005; accepted 30 May 2006

KEYWORDS Summary Chloroplasts; The electric and structural properties of envelope membranes of chloroplasts Envelope obtained from vegetative and generative plants of rape and the effect of hormone membranes; (IAA, GA and zearalenone) treatment were determined by zeta potential and small- Hormones; 3 angle X-ray scattering (SAXS) methods. Chloroplasts were isolated from leaves cut X-ray scattering; off from the vegetative (before cooling) and generative apical parts of plants. The Zeta potential lipid composition of chloroplast envelope membranes were analyzed by chromato- graphic techniques. Envelopes from generative plants contained higher levels of digalactosyldiacylglycerol (DGDG) and smaller amounts of phospholipids (PLs) in comparison to those obtained from vegetative ones. Moreover, envelopes of generative plants were characterized by higher fractions of unsaturated fatty acids. The zeta potential changes caused by hormone treatment were higher for chloroplasts isolated from vegetative plants in comparison to chloroplasts isolated from generative ones. An especially strong effect was observed for chloroplasts treated with IAA. The thickness of bilayers of untreated chloroplasts from vegetative plants were larger by 0.4 nm when comparing to the thickness of layers obtained from generative ones. The effect of hormones (GA3 and zearalenone) was detected only for vegetative chloroplasts.

ÃCorresponding author. Fax: +48 12 425 32 02. E-mail address: mariaﬁ[email protected] (M. Filek).

862 M. Filek et al.

Introduction Mannock and McElhaney, 2004). Thus, modifications of the chloroplast membrane composition related to the maintenance of optimal membrane functions The transformation of plants from vegetative to seem to be important also during the generative generative stage during development is associated development of winter plants (which needs peri- with several physiological events. The light and odic chilling for flowering induction). temperature requirements necessary for the flow- The reorganization of membrane lipids occurring ering induction of winter plants indicate that during generative development can affect the chloroplasts and their membranes can play an formation of specific domains important to hor- essential role in generative development (Possi- mone interactions. Hormones, especially GA and ngham, 1980; Miyamura et al., 1987; Masle, 2000; 3 IAA, play a significant role during generative Samala et al., 1998). development. Endogenous GA is essential in the Chloroplast membranes can be divided into two 3 photoperiodic control of grass flowering (Junttila et functional parts: envelope and thylakoid mem- al., 1997). Moreover, transport of IAA from apex to branes. The first are formed of two achlorophyllous roots is necessary for bud formation (Lomax et al., membranes, which separate the cytosol from the 1995). As it was presented in the literature plastid compartments. The thylakoid membranes (Kulaeva et al., 2000; Hole and Dodge, 1972; Misra contain chlorophyll and take a key role in photo- and Biswal, 1980; Chaudhry and Hussain, 2001), synthesis. Chloroplast envelopes and thylakoids are hormones participate in the control of chlorophyll composed of proteins and lipids with galactolipids: synthesis and its degradation during chloroplast monogalactosyldiacylglycerol (MGDG) and digalac- aging (in vivo and in vitro). tosyl-diacylglycerol (DGDG) (Moreau et al., 1998)as The aim of the paper was to determine the the dominant components. Therefore, the compo- electric and structural properties of chloroplast sition of chloroplast membranes is different from envelope membranes isolated from vegetative and the composition of other membranes containing generative winter rape and their changes caused by phospholipids (PLs) as a major lipid fraction. Both interaction with hormones of anionic type: GA , IAA MGDG and DGDG are uncharged. In a different way 3 and non-ionic zearalenone (Meng et al., 1996). The from DGDG and from the majority of membrane studies are concentrated on the properties of lipids which form bilayers (Simidjiev et al., 1998; envelope membranes of intact chloroplasts. Chlor- Lee, 2000) MGDG build-up non-bilayer structures. A oplasts were isolated from vegetative and gen- negative charge of chloroplast membranes is erative winter rape leaves which have a different related to the presence of sulfo-quinovosyldiacyl- morphological shape in these developmental glycerol (SQDG) and phosphatidylglycerol (PG). In stages. Comparison of electric and structural addition, zwitterionic phosphatidyl choline (PC) is changes of chloroplasts obtained from generative located in the envelope membranes (Moreau et al., and vegetative plants seems to be important in 1998). explaining the role of these organelles in the The unusual chemical composition of chloroplast mechanism of flowering induction. lipids is responsible for their unusual packing and the formation of specific domains which can be important for interaction between lipids and membrane proteins (Zaitsev et al., 1999) as well Material and methods as with other molecules adsorbed from the water phase (Shao et al., 1999; Tazi et al., 1999). It has Plant material been suggested that cell surface galactolipids serve as recognition sites for counterpart lectins and cell Seedlings of Brassica napus L. var. oleifera cv. adhesion receptors (Hakomort and Igarashi, 1995). Go´rczan´ski (winter) and cv. Mlochowski (spring) High levels of polyunsaturated galactolipids in were grown for 80 days in a controlled environment chloroplast membranes have been correlated with under a 16-h photoperiod (irradiance of the maintenance of membrane fluidity and with the 250 mmol m2 s1) and at day/night temperatures prevention of chilling injuries (Welburn, 1997; of 20/17 1C. Then, the plants were cooled to 5/2 1C ARTICLE IN PRESS

Electric and structural studies of hormone interaction 863

(vernalization) for 63 days under the same condi- Small-angle X-ray scattering (SAXS) tions of day length and irradiance. Following the experiments cold treatment, the plants were grown at 20/17 1C until the appearance of generative leaves. Apical The intact chloroplast structure was determined segments that were 15 mm long including the from SAXS measurements carried out on a SWAX- youngest leaf of 10–20 mm long were cut off from camera (HECUS X-ray Systems, Graz, Austria) as the vegetative (before cooling) and generative described previously (Laggner and Mio, 1992; plants and placed on the MS medium (Murashige Laggner, 1994; Filek et al., 2005). Ni-filtered 3 and Skoog, 1962), which contained 0.1 mg dm Cu–Ka-radiation (l ¼ 1:54 A)˚ originating from a benzylaminopurine (BAP) and 3% sucrose. Under rotating Cu-anode X-ray generator, (Rigaku, Japan) these conditions the plants were cultured during 1 operating at 50 kV and 60 mA was used. SAXS angle week at day/night temperatures of 20/171 with a calibration was done with silver stearate. Chlor- 2 1 16-h photoperiod (irradiance of 250 mmol m s ). oplasts were used in a concentration of 100 mg Plants prepared this way were used for chloroplast chloroplast lipids/ml in CIB and the measurements isolation. were performed with samples placed in a 1-mm quartz capillary at 20 1C with an exposure time of 1800 s. The SAXS curves were analyzed after back- Chloroplast isolation ground subtraction and normalization in terms of distance distribution functions p(r) derived by Intact chloroplasts were isolated from fresh leaf indirect Fourier transformation (Laggner, 1988). tissues by the method Block et al. (1983) in chloroplast isolation buffer (CIB), containing Lipid and fatty acids analysis 50 mM Tris–HCl, 5 mM EDTA, 0.33 M sorbitol, pH 7.5 and separated from broken chloroplasts by Chloroplast envelopes were separated from centrifugation in a 40%/80% Percoll gradient. The thylakoid membranes according to Poicelot’s chloroplast were resuspended in CIB and, until (1977) method. Envelope membranes were homo- further use, kept on ice in the dark. The measure- genized in a mixture of chlorofom: isopropanol ments were performed within a time period not (1:1, v/v) and lipids were re-extracted with chloro- longer than 0.5 h after preparation. As is found in form (Bligh and Dyer, 1959). Glycolipids (MGDG and the literature (Polanska´ et al., 2004) and confirmed DGDG) and PL fractions were isolated using by our own observations, during this time the adsorptive and distributive column chromatography interval chloroplasts stayed intact in the sense of on silica acid under low nitrogen pressure. The keeping unchanged shape (checked under micro- purity of polar fractions was checked by thin-layer scope) and constant conductivity of the suspension. chromatography (Block et al., 1983). Individual lipid classes (MGDG, DGDG and PL) were trans- Zeta potential measurements methylated with BF3 (14% in methanol). The obtained methyl esters were then quantified by gas chromatography (Hewlett Packard, USA) with a The zeta potential values (z) were determined capillary column (30 m 0.25 mm) at 170 1C. The from electrophoretic mobility data measured with identification of fatty acids was performed using Zeta-PLUS apparatus (Brookhaven, USA). The pal- appropriate standards. The quantification of fatty ladium electrodes were used for an electric field acid content was carried out using 17:0 acid as the application. Electric conductance was determined internal standard. simultaneously for each measurement. The zeta potential measurements of chloroplasts were performed in media of defined ionic composition and ionic strength at 20 1C. The stock supporting Results electrolyte contained: 1 mM KCl, 0.3 mM NaCl and 1 mM MES–KOH buffer (pH 5.6) plus 0.6 mM manni- The composition of chloroplast envelope lipids tol. Indole-3-acetic acid (IAA), gibberellic acid indicated that generative development of rape (GA3) or zearalenone (6-(10-hydroxy-6-okso-trans- plants is accompanied by an increase in the level of 1-undecenyl)-benzoic acid lactone) were intro- both galactolipids MGDG and DGDG while there is a duced to the supporting electrolyte at the constant decrease in the PL content (Table 1). Linolenic acid weight concentration equal to 25 mg dm3, which (18:3) was the major fatty acid of all lipids in both corresponds to 143, 72 and 78.5 mM for IAA, GA3 and developmental stages. However, the unsaturation zearalenone, respectively. degree of DGDG and of PL, calculated as a ratio of ARTICLE IN PRESS

864 M. Filek et al.

Table 1. Distribution of fatty acids among the polar lipids of chloroplast envelopes isolated from vegetative and generative winter rape

Object Lipid fractions (mol% of polar lipids) Fatty acids (mol%)

16:0 18:0 18:1 18:2 18:3 18:3/18:2

Vegetative MGDG 43.270.5 4.770.1 0.570.1 28.570.4 6.070.2 60.270.3 10.070.3 DGDG 16.770.9 19.470.3 2.270.3 6.870.2 6.270.1 65.370.4 10.570.4 PL 39.970.8 34.270.4 3.570.8 7.570.5 13.870.3 41.070.1 3.070.3 Generative MGDG 45.570.6 6.970.1 0.570.1 25.170.1 6.570.2 60.870.2 9.370.3 DGDG 18.470.9 11.970.2 1.370.1 12.770.5 4.670.2 69.570.5 15.170.4 PL 35.970.9 29.870.3 3.570.2 8.170.3 9.270.1 49.570.3 5.470.2

Note: The data are averages of three independent replications 7SE MGDG, monogalactosyldiacylglycerols; DGDG, digalactosyldiacylglycerols; PL, phospholipids.

Figure 1. Zeta potential (z) of intact chloroplasts isolated from rape leaves measured as a function of Figure 2. The absolute values of z potential of chlor- medium conductance (1/R). The medium conductance oplasts isolated from vegetative and generative leaves of was changed by dilution of the chloroplast isolation rape in media without (0) and with hormones: IAA, GA3 or buffer (CIB) with water. The points are averages from 30 zearalenone. Measured medium contained: 1 mM KCl, measurements 7SE. 0.3 mM NaCl and 1 mM MES–KOH buffer (pH 5.6) plus 0.6 mM mannitol. Hormones were introduced to the supporting medium at the constant concentration equal 3 18:3/18:2 in a generative stage was higher than the to 25 mg dm . Results are average from 30 measure- ratio obtained in a vegetative one. By contrast, the ments 7SE. unsaturation degree of MGDG was constant in both developmental stages with small differences no- ticed in 16:0 and 18:1 acids only. Traces of 16:1 buffer). Under these conditions, z potential values fatty acid were detected in the DGDG fraction. for chloroplasts isolated from vegetative and To choose appropriate conditions for the char- generative plants were equal to 20.271.2 and acterization of the electric properties of chloro- 17.870.4 mV, respectively (Fig. 2) and they were plast envelope membranes, the zeta potential (z) constant for about 5 h. These z potential values for of intact chloroplasts was measured as a function of reference chloroplasts were large enough to notice suspension conductance (Fig. 1). The suspension changes caused by hormones. conductance was changed by dilution of CIB with The absolute values of the z potential of water. To determine zeta potential changes caused chloroplasts isolated from vegetative and genera- by hormone interaction, the CIB concentration of tive plants after applying all investigated hormones conductance of about 600 mS was chosen (which decreased (Fig. 2). The largest change was corresponds to 20 times dilution of the stock detected for IAA (despite the fact that its molar ARTICLE IN PRESS

Electric and structural studies of hormone interaction 865 concentration was approximately two times lower long-range order) bilayer lipid membranes with a than for the other hormones studied) and the visible maximum reﬂecting a certain bilayer thick- smallest one for zearalenone. The effect of IAA was ness (Fig. 3). The noticeable shift in the angular especially visible in the case of chloroplasts position of the maximum was observed when obtained from vegetative plants for which the z comparing results obtained for both investigated potential changed by about 15 mV, whereas in the chloroplasts. Structural parameters in terms of the case of generative ones, the change in the z lamellar thickness of the lipid/water system were potential amounted to only about 7 mV. For derived from the continuous Fourier transformation chloroplasts from vegetative and generative plants of the SAXS curves (Table 2). treated by GA3, the same values of z potential were The difference between bilayer thicknesses measured. However, when comparing to reference obtained for chloroplasts of vegetative and gen- non-treated organelles the relative effect of this erative origin was about 0.4 nm. The treatment of hormone was higher for vegetative membranes. chloroplasts of generative plants with all hormones The relative effect of zearalenone on chloroplasts investigated did not affect the bilayer thickness. of both types was similar. However, in the case of chloroplasts of vegetative The SAXS measurements for the chloroplasts origin, a small decrease in bilayer thicknesses was studied resulted in continuous scattering curves detected after treatment with GA3 and zearale- characteristic for uncorrelated (with no regular none whereas IAA did not cause any change in the value of this parameter (Table 2).

Discussion

The lipid and fatty acid composition of chloroplast envelope membranes of vegetative and generative rape is typical of plant chloroplasts and it is well represented by the data of Moreau et al. (1998). However, observed differences in lipid and fatty acid proportions (i.e., a decrease in the PL fraction and an increase in the fatty acid unsaturation degree) between generative and vegetative organelles can be connected with the generative development of a plant. The negative charge of the investigated chloroplasts is connected with the presence of PLs in the envelope membranes. The various structural and functional features of the membranes can be Figure 3. Experimental SAXS scattering curves. The correlated with their surface potential (Kraayenhof graph represents the dependence of scattered intensity ( 1 et al., 1996). The charged interface facilitates the (arbitrary units) vs. reciprocal scattering vector q (A ), q ¼ð4p=lÞsin y , (where l is the wavelength of X-ray, and formation of a hydration layer of oriented water 2y is the scattering angle) for chloroplasts isolated from molecules that may stabilize the lipid head-group vegetative and generative leaves of rape (100 mg/ml of region (Kraayenhof et al., 1996). In our earlier CIB buffer) after background subtraction (scattering of studies on wheat membranes, it was shown that the buffer solution). The two curves are shifted vertically negatively charged plasmalemma in wheat cells can for clarity. adsorb negatively and positively charged hormones

Table 2. Head-to-head group thickness of the lipid bilayer in average for all types chloroplast membranes after hormone (IAA, GA3, zearalenone, 0 – without hormones) treatment, calculated from SAXS data for envelope chloroplasts obtained from vegetative and generative winter rape

Chloroplasts Membrane thickness (nm)

0 IAA GA3 Zearalenone

Vegetative 3.9370.10 3.9470.11 3.7170.13 3.7070.11 Generative 3.5270.10 3.5370.10 3.5070.12 3.4970.11 ARTICLE IN PRESS

866 M. Filek et al. as well as non-ionic zearalenone (Filek et al., branes results in the occurrence of low hydrated 2002). This effect was discussed in terms of the ‘‘pores’’ (Moreau et al., 1998). interaction between the hormones investigated and The bilayer thicknesses determined in SAXS the PL and protein domains. experiments are larger for all types of membranes The results presented here show that the treat- present in chloroplasts isolated from vegetative ment of rape chloroplast envelope membranes with plants in comparison to those of generative origin. the hormones studied led to a noticeable decrease This observation correlates well with composition in absolute values of zeta potentials. The larger of envelope membranes i.e. unsaturation degree effect of negatively charged IAA and GA3 in and MDGD content. However, one has to mention comparison to non-ionic zearalenone can be re- that SAXS technique measures all membranes lated to the interaction of hormones with zwitter- present in the system giving information about ionic PC, which represents a major fraction of the the weighted sum of all thicknesses of the PLs present in a chloroplast envelope. This can also membranes. The signal from the membranes is explain the weaker effect of all hormones on the weighted by their contrast (with respect to the zeta potential of chloroplasts of generative plants buffer) and their volume. The contrast is most (whose membranes contain less PC in comparison to likely the same for all types of membranes. Those vegetative plants). The hormones studied can also membranes which are quantitatively dominating interact with proteins present in membranes. (by number and volume) will contribute most to the However, taking into account the smaller content scattering signal. of proteins in envelope membranes (in comparison The experiments presented indicate that the to plasmalemma as studied before (Filek et al., changes in the composition of chloroplast envelope 2002)) as well as a comparable level of proteins in membrane lipids during generative development both types of chloroplasts (data not indicated), one lead to changes in electric and structural proper- can relate the changes in zeta potential as being ties. These changes can also affect the adsorption caused predominantly by the interaction of hor- of ionic and non-ionic hormones. The bilayer mones with lipids. Nevertheless, some contribution thickness of membranes of generative objects was to these changes can be related to differences in not affected by the hormones studied whereas this protein hormone receptors characteristic for chlor- parameter for vegetative membranes decreased oplast envelopes of vegetative and generative after interaction with GA3 and zearalenone. The origin. electric properties of vegetative chloroplasts were The changes in zeta potential of hormone- affected to a higher degree by treatment with treated membranes can be interpreted in terms hormones (especially IAA and GA3) in comparison to of hormone adsorption. The higher changes in zeta generative objects. The results presented show potential obtained for envelopes of vegetative that zeta potentials more than structural para- plants (especially large after treatment with IAA) meters are sensitive to membrane surface modifi- in comparison to those of generative ones may be cations caused by hormone treatment. This last associated with a larger amount of adsorbed observation is related to the fact that electro- hormones. The treatment of chloroplasts of vege- kinetic parameters are determined by the outmost tative and generative origin by zearalenone did not surface of the object whereas SAXS signal contains produce noticeable changes in zeta potentials, information about all membranes present in the indicating that adsorption of this hormone, if any, system. does not affect the electric state of the membranes. Modification of envelope composition can be Acknowledgement accompanied with structural changes in the chloroplast membranes of vegetative and generative This work was partially supported by the MEiN plants. In spite of the many observations made on grant No. 1 T09A 122 30. model membranes containing defined lipids occurring separately or in mixtures, little is known about the structural organization and properties of References natural membranes. The bilayer thickness of model membranes is decreasing with an increase of Bligh EG, Dyer WJ. A rapid method of total lipids content of unsaturated fatty acids (Rawicz et al., extraction and purification. Can J Biochem 1959;37: 2001). Also a higher level of MGDG facilitates the 911–5. formation of thinner bilayers, MGDG itself forms Block AM, Dorne AJ, Joyard J, Douce R. Preparation and non-lamellar structures and its presence in mem- characterization of membrane fractions enriched in ARTICLE IN PRESS

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outer and inner envelope membranes from spinach anatomy in young wheat plants are modulated by chloroplasts. J Biol Chem 1983;258:13281–6. factors related to leaf position, vernalization, and Chaudhry NY, Hussain UK. Effect of growth hormones i.e. genotype. Plant Physiol 2000;122:1399–415. GA3, IAA and kinetin on the external morphology and Meng F-J, Han Y-Z, Fu Y-F, Guo F-L. Zearalenone in higher flowering of Phaseolus aureus L. J Biol Sci 2001;1: plants. FNL 1996;22:54–7. 801–4. Misra AN, Biswal UC. Effect of phytohormones on Filek M, Gzyl B, Laggner P, Kriechbaum M. Effect of chlorophyll degradation during aging of chloroplasts indole-3-acetic acid on surface properties of the in vivo and in vitro. Protoplasma 1980;105:1–5. wheat plastid lipids. J Plant Physiol 2005;162:45–252. Miyamura S, Kuroiwa T, Nagata T. Disappearance of plastid Filek M, Zembala M, Szechyn´ska-Hebda M. The influence and mitochondrial nucleoids during the formation of phytohormones on zeta potential and electrokinetic of generative cells of higher plants revealed by charges of winter wheat cells. Z Naturforsch 2002;57c: fluorescence microscopy. Protoplasma 1987;141: 696–704. 149–59. Hakomort S, Igarashi Y. Functional role of glycosphingo- Moreau P, Bessoule JJ, Mongrand S, Testet E, Vincent P, lipids in cell recognition and signalling. J Biochem Cassagne C. Lipid trafficking in plant cells. Prog Lipid 1995;118:1091–103. Res 1998;37:371–91. Hole CC, Dodge AD. The interaction of a gibberellin and Murashige T, Skoog F. Revised medium for rapid growth kinetin in the control of chlorophyll synthesis. Cell and bioassay with tobacco issue cultures. Physiol Plant Moll Life Sci 1972;28:1113–4. 1962;15:473–9. Junttila O, Heide OM, Lindgard B, Ernstsen A. Gibber- Poincelot R. Isolation of envelope membranes from ellins and photoperiodic control of leaf growth in Poa bundle sheath chloroplasts of maize. Plant Physiol pratensis. Physiol Plant 1997;101:599–605. 1977;60:767–70. Kraayenhof R, Sterk GJ, Wong Fong Sang HW, Krab K, Epand Polanska´L,Vicˇańkova´ A, Dobrev PI, MachaćˇkovaÍ, RM. Monovalent cations differentially affect membrane Vanˇkova´ R. Viability, ultrastructure and cytokinin surface properties and membrane curvature, as re- metabolism of free and immobilized tobacco chlor- vealed by fluorescent probes and dynamic light scatter- oplasts. Biotech Lett 2004;26:1549–55. ing. Biochim Biophys Acta 1996;1282:293–302. Possingham JV. Plastid replication and development in Kulaeva ON, Karavaiko NN, Selivankina Syu, Kusnetsov VV, the life cycle of higher plants. Ann Rev Plant Physiol Zemlyachenko YaV, Cherepneva GN, et al. Nuclear and 1980;31:113–29. chloroplast cytokinin-binding proteins from barley Rawicz W, Olbrich KC, McIntosh T, Needham D, Evans E. leaves participating in transcription regulation. Plant Effect of chain length and unsaturation on elasticity of Growth Reg 2000;32:329–35. lipid bilayers. Biophys J 2001;79:328–39. Laggner P. X-ray studies on biological membranes using Samala S, Yan J, Baird V. Changes in polar lipid fatty acid synchrotron radiation. Top Curr Chem 1988;145: composition during cold acclimation in ‘Midiron’ and 175–200. ‘U3’ Bermudagrass. Cep Sci 1998;38:188–95. Laggner P. X-ray diffraction on biomembranes with Shao L, Konka V, Leblanc RM. Surface chemistry studies of emphasis on lipid moiety. Subcell Biochem 1994;23: photosystem II. J Coll Interf Sci 1999;215:92–8. 451–91. Simidjiev I, Barzda V, Mustardy L, Garab G. Role of Laggner P, Mio H. SWAX – a dual-detector camera for thylakod lipids in the structure flexibility of lamellar simulations small- and wide-angle X-ray diffraction in aggregates of the isolated light-harvesting chlorophyll polymer and liquid crystal research. Nucl Instrum a/b complex of photosystem II. Biochem 1998;37: Methods Phys Res 1992;A323:86–90. 4169–73. Lee AG. Membrane lipids: it’s only a phase. Curr Biol Tazi A, Boussaad S, Leblanc RM. Atomic force microscopy 2000;10:377–80. study of cytochrome f (Cyt f) and mixed monogalac- Lomax TI, Muday GK, Rubery P.Auxin transport. In: Davies tosyldiacylglycerol (MGDG)/Cyt f Langmuir–Blodget PJ, editor. Plant hormones: physiology, biochemistry, films. Thin Solid Films 1999;353:233–8. and molecular biology. Dordrecht, The Netherlands: Welburn AR. Environmental factors and genotypic Kulwer Academic Publishers; 1995. p. 509–30. changes in lipids contribute to winter hardiness of Mannock DA, McElhaney RN. Thermotropic and lyotropic Norway spruce (Picea abies). New Phytobiol 1997;135: phase properties of glycolipid diastereomers: role of 115–21. headgroup and interfacial interactions in determining Zaitsev SY, Kalabina NA, Herrmann B, Schaefer C, Zubov phase behavior. Curr Opt Coll Interf Sci 2004;8:426–47. VP. A comparative study of the photosystem II Masle J. The effects of elevated CO2 concentrations on membrane proteins with natural lipids in monolayers. cell division rates, growth patterns, and blade Mater Sci Imag 1999;8:519–22.