Mutants of Saccharomyces cerevisiae cell division cycle defective in cytokinesis. Biosynthesis of the cell wall and morphology

The four temperature-sensitive mutants of Saccharomyces cerevisiae in the cell division cycle defective in cytokinesis (cdc, 3, 10, 11 and 12), have been analyzed with respect to the biosynthesis of the cell wall polymers. After 3 hours of incubation at the non-permissive temperature (37°C) these strains stop growing. The synthesis of glucan, mannan and chitin (wall polymers) level off in a similar time, but glucan, mannan and chitin synthases remained active for at least 4 hours.If the mutants are analyzed by transmission and scanning electron microscopy different pictures emerge. Two of the mutants cdc 10 and cdc 12, after 3 hours of incubation at 37°C present apparently normal cytoplasms and cell wall surfaces with multiple elongated buds. The other two mutants, cdc 3 and cdc 11, present a completely disarranged cytoplasmic content and damage at the level of the plasma membrane is evident.These and other observations, suggest that between the execution points of cdc 3 (0.27) and cdc 10 (0.58), essential processes in the assembly of cell membrane occur.This work was supported in part by a grant from la Comisión de Investigación Científica y Técnica of the Spanish Ministerio de Educación y Ciencia (Project no. 4593-1980).     

Decreased mitochondrial biogenesis in temperature-sensitive cell division cycle mutants of Saccharomyces cerevisiae

The temperature-sensitive cell division cycle (cdc) G1 mutants cdc28 and cdc35 show decreased mitochondrial volumes with respect to the wild type strain A364A (WT) at the restrictive temperature. Of the three criteria of mitochondrial biogenesis studied, that is, number of mitochondria per cell, relative area of the cell occupied by mitochondria, or relative area of mitochondria occupied by inner membranes, only the second indicator was significantly lower in cdc mutants than in the WT. The mitochondrial inner membranes development did not compensate for the decrease in the organelles volume. Apparently, the reduced mitochondrial biogenesis was not due to the temperature shift because the relative area of the cell occupied by mitochondria was already significantly lower at 25°C in cdc mutants. The specific fluxes of oxygen consumption confirmed that the respiratory capacity of cdc mutants is largely impaired in respect to the WT. Cdc28 and cdc35 mutants of Saccharomyces cerevisiae had been previously shown to exhibit high respiratory quotients (from 3 to 7) in respect to the WT (RQ 1.0), which correlated with carbon and energy uncoupling probably the result of glucose-induced catabolite repression [Aon MA, Mónaco ME, Cortassa S (1995) Exp Cell Res 217, 42–51; Mónaco ME, Valdecantos PA, Aon MA (1995) Exp Cell Res 217, 52–56].     

SelectingRhizobium phaseoli strains for use with beans (Phaseolus vulgaris L.) in Kenya: Tolerance of high temperature and antibiotic resistance

Forty one strains ofRhizobium phaseoli were screened for the ability to multiply at high temperatures on yeast extract-mannitol agar. Most strains were tolerant of 30°C, eight strains were tolerant of 45°C and two of 47°C although the rate of multiplication was reduced at 45–47°C. The high temperature-tolerant strains were isolated from Kenyan soils and were fast-growing. Seven of the eight strains tolerant of 45–47°C lost their infectiveness after incubation at high temperature but four strains tolerant of 40°C remained infective after incubation at that temperature.Thirty six strains were resistant to 200 g ml^–1 streptomycin sulphate and 29 strains to 200 g ml^–1 spectinomycin dihydrochloride. Eight strains were resistant to both antibiotics each at 200 g ml^–1. Two of the double-labelled antibiotic-resistant mutants lost their infectiveness onPhaseolus vulgaris. The response to acidity was unaltered and two of the mutants showed a decrease in temperature tolerance. The doublelabelled mutants were recoverable from two Kenyan soils.     

Mutants with reduced Ca activation inParamecium aurelia

Summary Two heat-sensitive pawn mutants ofParamecium aurelia are capable of avoiding reactions when grown at 23°C but not at 35°C. Electrophysiological analyses show that Ca activation is reduced in the mutants even when they are grown at 23°C. The maximal rate of rise and the peak of the evoked action potential (Ca-spike) in the mutants are smaller than those of wild type in a K-solution. After suppression of K conductance by either TEA⁺ or Ba⁺⁺, the action potentials of the mutants peak at the same level as that of wild type. However, the maximal rate of rise of the mutants remains only about half that of wild type. Thus, the mutations affect Ca activation but not K activation.Incubation at a high temperature (35°C) further reduces Ca activation to almost zero in the mutants but has little or no effect on wild type. This almost complete loss of Ca activation explains the lack of avoiding reactions when the mutants are grown at high temperatures. A double mutant containing two heat-sensitive mutations shows extremely reduced Ca activation even when grown at 23°C.     

Effects on growth and sporulation of inactivation of a Bacillus subtilis gene (ctc) transcribed in vitro by minor vegetative cell RNA polymerases (E-sigma 37, E-sigma 32)

Summary The E-³⁷ gene ctc was inactivated by a site-specific insertion into the Bacillus subtilis chromosome. The resulting mutation inhibited sporulation by 95% at elevated temperatures (48° C). If the ctc ^- mutation is placed in a strain that carries a mutation in the closely linked but distinct spoVC gene, ctc now affects both growth and sporulation at elevated temperatures. Growth of the ctc ^- spoVC285 strain was transiently inhibited when exponentially growing cultures were shifted from 37° C to 48° C. A similar, but less pronounced growth lag, was also seen in a B. subtilis strain carrying only the spoVC-285 mutation. This finding suggests that both the ctc and spoVC products function in vegetatively growing B. subtilis.     

Influence of muskmelon cell wall polysaccharides on roridin E production by a pathogenic strain of Myrothecium roridum

Addition of fruit cell wall extracts from two muskmelon cultivars into liquid media affected mycotoxin production by a strain of Myrothecium roridum pathogenic to muskmelon. Cell wall extracts from a susceptible cultivar (Iroquois) significantly increased toxin production while cell wall extracts from a resistant cultivar (Hales Best) significantly inhibited toxin production. Media containing 0.1 or 1.0 mg ml^–1 stimulated toxin production more than media containing 10 or 100 mg ml^–1 of cell wall extracts. Previous studies in our laboratory suggest that roridin E may be involved in virulence or pathogenicity of M. roridum; the present study indicates that cell wall polysaccharides as well as other materials present in cell wall preparations from susceptible host tissue provide a better substrate for toxin production than cell wall preparation from resistant host tissue.     

Temporal and spatial changes of cellulose synthesis inClosterium acerosum (Schrank) Ehrenberg during cell growth

The amount and distribution of wall microfibril synthesis were investigated in the cell-division cycle ofClosterium acerosum. Electron-microscopic examination and a methylation analysis of alkali-extracted wall fragments showed that alkali-extracted wall was mainly composed of microfibrils and that the microfibrils ofC. acerosum were 4-linked glucans, i.e., cellulose. Cellulose synthesis was measured as incorporation of¹⁴C, fed to cells as NaHCO₃, into extracted wall fragments. Extensive cellulose synthesis was coincident with septum formation, continued for more than 6 h and then ceased. It was found by microautoradiography that cellulose synthesis after cell division was essentially restricted to the expanding new semicells. Such a restricted distribution of cellulose synthesis was maintained for more than 6 h after septum formation, i.e., for more than 2 h after the cessation of expansion; afterwards, cellulose synthesis in some, but not all, cells became extended to the old semicells, and then ceased. Considerable cellulose synthesis also took place in the band-like expanding part of non-divided cells, indicating that cell division was not necessarily required for the induction of cellulose synthesis and the latter was coupled with cell expansion. Extension of cellulose synthesis to old semicells was brought about in divided cells by treatment with 3 mM colchicine, 28 M vinblastine, 50 M isopropyl-N-phenylcarbamate or 1 M isopropyl-N(3-chlorophenyl)carbamate, indicating that microtubules are involved in the limitation of cellulose synthesis to the new semicells.Abbreviations CIPC isopropyl-N(3-chlorophenyl)carbamate - DPO 2,5-diphenyloxazole - IPC isopropyl-N-phenylcarbamate     

Quantitative aspects of RNA synthesis in normal and lithium-treated embryos ofXenopus laevis

Summary The treatment ofXenopus early embryos with lithium chloride produces exogastruale — embryos which fail to gastrulate normally and in which the rates of cell division are reduced. In the present study estimations of incorporations of (5-³H) uridine and the specific activities of the 5-ribonucleotide precursor pools showed that exogastrulae have higher rates of RNA synthesis per cell than control neurulae. Sub-cellular fractionations showed that a greater proportion of labelled RNA was retained in the nuclei of exogastrulae than of neurulae, while neurulae showed a greater incorporation into polysomes.     

Effects of temperature on cytomorphogenesis and ultrastructure ofMicrasterias denticulata Bréb

Summary Exposure of growingMicrasterias cells to high (32°–36°C) and low (3°–10°C) temperatures produces changes in morphology that are accompanied by several ultrastructural alterations. Whereas low temperatures essentially cause simplification of cell ornamentation, a variety of cell malformations result from high temperature treatment. These are the loss of cell symmetry leading to markedly aberrant cell shapes and an increase of main lobes with reduced degree of differentiation. Preliminary studies indicate that a shift in the distribution of membrane-associated Ca²⁺ by elevated temperatures probably underlies these abnormal cytomorphogenetic events. Both, low and high temperature cause a reduction in size of the young half cell and affect cytoplasmic streaming. Moreover, nuclear migration is retarded and chloroplast arrangement is influenced by temperature treatment at both ranges. Growth velocity of primary wall responsible for cell shaping is increased at high and slowed down at low temperatures compared to cells grown at 20°C.The main ultrastructural alterations induced by high temperatures are an increase in amount and length of ER cisternae, the appearance of heat shock granule aggregations localized in the cytoplasm, a reduced number of ribosomes and polysomes, the presence of oil bodies in growing cells and a varying thickness of the primary wall. Influences of low temperatures on ultrastructure are less pronounced. They are manifested in the formation of large aggregations of ER cisternae slightly differing from those found in untreated cells, a disturbed arrangement of the microtubule system surrounding the nucleus and a decrease of the number of cell wall forming cytoplasmic vesicles.It is thought that most of the temperature effects are due to an influence on membranes probably an alteration of ionic currents and, in addition, a modulation of normal protein synthesis.Dedicated to my teacher Professor Oswald Kiermayer in deep gratitude     

The function of chitin synthases 2 and 3 in the Saccharomyces cerevisiae cell cycle

The morphology of three Saccharomyces cerevisiae strains, all lacking chitin synthase 1 (Chs1) and two of them deficient in either Chs3 (calR1 mutation) or Chs2 was observed by light and electron microscopy. Cells deficient in Chs2 showed clumpy growth and aberrant shape and size. Their septa were very thick; the primary septum was absent. Staining with WGA-gold complexes revealed a diffuse distribution of chitin in the septum, whereas chitin was normally located at the neck between mother cell and bud and in the wall of mother cells. Strains deficient in Chs3 exhibited minor abnormalities in budding pattern and shape. Their septa were thin and trilaminar. Staining for chitin revealed a thin line of the polysaccharide along the primary septum; no chitin was present elsewhere in the wall. Therefore, Chs2 is specific for primary septum formation, whereas Chs3 is responsible for chitin in the ring at bud emergence and in the cell wall. Chs3 is also required for chitin synthesized in the presence of alpha-pheromone or deposited in the cell wall of cdc mutants at nonpermissive temperature, and for chitosan in spore walls. Genetic evidence indicated that a mutant lacking all three chitin synthases was inviable; this was confirmed by constructing a triple mutant rescued by a plasmid carrying a CHS2 gene under control of a GAL1 promoter. Transfer of the mutant from galactose to glucose resulted in cell division arrest followed by cell death. We conclude that some chitin synthesis is essential for viability of yeast cells.     

The localization of pentosans within the cell wall of aspen (Populus tremuloides Michx.) by high resolution autoradiography

Summary Radiochemical studies of Populus tremuloides xylem tissue administered l-[1-³H]arabinose, d-[1-³H]glucose, and d-[6-³H]glucose demonstrate that l-[1-³H]arabinose is an excellent precursor for pentosan in this tissue. Transverse sections of first-year xylem (from cambial zone to pith) were examined by light and electron microscope autoradiography. Relatively large amounts of labeled pentosan are found in parenchyma cell walls, including the protective layer of ray parenchyma. Computeraided analyses of grain distributions in electron micrographs of cell walls of individual fibers localized the labeled wall components after different periods of incubation by comparison to model behavior. These analyses indicate that pentosan is added to the secondary cell wall of developing fibers by an appositional mechanism.     

Production of β-fructofuranosidase byAspergillus japonicus

A newly isolated strain, MU-2, which produces very high -fructofuranosidase activity, was identified asAspergillus japonicus. For enzyme production by the strain, sucrose at 20% (w/v) was the best carbon source and yeast extract at 1.5 to 3% (w/v) the best nitrogen source. Total enzymatic activity and cell growth were at maximum after 48 h, at 1.57×10⁴ U/flask and 0.81 g dry cells/flask, respectively. The optimum pH value of the enzymatic reaction was between 5.0 and 5.5 and the optimum temperature 60 to 65°C. The enzyme produced 1-kestose (O--d-fructofuranosyl-(21)--d-fructofuranosyl -d-glucopyranoside) and nystose (O--d-fructofuranosyl-(21)--d-fructofuranosyl-(21)--d-fructofuranosyl -d-glucopyranoside) from sucrose by fructosyl-transferring activity. The strain was found to be very useful for industrial production of -fructofuranosidase.     

Temperature regulation of oleate desaturase in sunflower (Helianthus annuus L.) seeds

The effect of temperature on oleate desaturation in developing sunflower (Helianthus annuus L.) seeds has been examined. When seeds from plants grown at low (20/10° C, day/night) temperature were transferred for 24 h to 10° C, an increase in the linoleate/oleate ratio in phosphatidylcholine and triacylglycerol was observed, but not when transfer was to 20 or 30° C. The same effect was observed in triacylglycerol, phosphatidylcholine and phosphatidylethanolamine in the newly synthesized lipids after in-vivo incubation with [1-¹⁴C]oleate at 10° C. The microsomal oleoyl phosphatidylcholine desaturase (ODS) activity of the seeds maintained at 10 C was also enhanced. The stimulation was observed after only 3 h in plants grown at high temperature (30/20° C). This effect was inhibited by cycloheximide, implying that the low-temperature stimulation of the ODS activity was caused by the synthesis of new enzyme. As a consequence, seeds from plants grown at low temperature had higher ODS activities and linoleate contents than those grown at high temperature. The microsomal ODS activity of seeds from plants grown at low temperature was dependent on incubation temperature and showed a maximum at 20° C. By contrast, this activity was almost temperature-insensitive in seeds from plants grown at high temperature. These results could explain how temperature regulates the fatty-acid composition in sunflower-seed lipids.Abbreviations DAF days after flowering - ODS oleoyl phosphatidylcholine desaturase - PC phosphatidylcholine - PE phosphatidylethanolamine - TAG triacylglycerol - 181 oleic acid - 182 linoleic acid To whom correspondence should be addressedThanks are due to M.C. Ruiz for skillful technical assistance. This work was supported by a grant from Junta de Andalucia, Spain.     

Elevated recombination and pairing structures during meiotic arrest in yeast of the nuclear division mutant cdc5

Summary A diploid strain of yeast, homozygous for the mutation cdc5-1, undergoes a normal meiosis at 25° C. At the nonpermissive temperature of 34° C, meiosis is arrested at the first meiotic division, after premeiotic DNA replication and recombination commitment have taken place. Haploidisation commitment does not occur at 34° C. Electron microscopy reveals that synaptons (synaptonemal complexes) are formed and the stage of arrest is characterised by a prevalence of modified synaptons, which consist of paired lateral elements lacking the central elements. Prolonged incubation at this stage of arrest results in unusually high recombination levels, perhaps related to the synaptonal structures observed.Temperature shift-up experiments (transfers of cells from 25° C to 34° C at various times during meiosis) reveal that the CDC5 function is required for both the first and the second divisions of meiosis.     

Characterization of long-term extension of isolated cell walls from growing cucumber hypocotyls

Walls from frozen-thawed cucumber (Cucumis sativus L.) hypocotyls extend for many hours when placed in tension under acidic conditions. This study examined whether such creep is a purely physical process dependent on wall viscoelasticity alone or whether enzymatic activities are needed to maintain wall extension. Chemical denaturants inhibited wall creep, some acting reversibly and others irreversibly. Brief (15 s) boiling in water irreversibly inhibited creep, as did pre-incubation with proteases. Creep exhibited a high Q₁₀ (3.8) between 20° and 30°C, with slow inactivation at higher temperatures, whereas the viscous flow of pectin solutions exhibited a much lower Q₁₀ (1.35). On the basis of its temperature sensitivity, involvement of pectic gel-sol transitions was judged to be of little importance in creep. Pre-incubation of walls in neutral pH irreversibly inactivated their ability to creep, with a half-time of about 40 min. At 1 mM, Cu²⁺, Hg²⁺ and Al³⁺ were strongly inhibitory whereas most other cations, including Ca²⁺, had little effect. Sulfhydryl-reducing agents strongly stimulated creep, apparently by stabilizing wall enzyme(s). The physical effects of these treatments on polymer interactions were examined by Instron and stress-relaxation analyses. Some treatments, such as pH and Cu²⁺, had significant effects on wall viscoelasticity, but others had little or no apparent effect, thus implicating an enzymatic creep mechanism. The results indicate that creep depends on relatively rugged enzymes that are firmly attached to or entangled in the wall. The sensitivity of creep to SH-reducing agents indicates that thiol reduction of wall enzymes might provide a control mechanism for endogenous cell growth.Abbreviations DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - EGTA ethyleneglycol-bis-(-aminoethylether)-N,N,N,N-tetraacetic acid - Hepes N-2-hydroxyethylpiperazine-N-2-ethansulfonic acid     

Immunofluorescence microscopy of microtubule arrangement in Closterium acerosum (Schrank) Ehrenberg

The microtubule (MT) arrangement in Closterium acerosum cells was observed by indirect immunofluorescence microscopy both during and following cell division, and during cell expansion without cell division. (During the division period, some cells of this alga divide whereas other cells expand in their middle region without division.) Before septum formation, all cells had a ring-like MT bundle (MT ring) in their middle. Both septum formation and expansion without cell division occurred at the position of this ring. During the periods of division, short, hair-like MTs appeared around the nucleus in some of the cells, in addition to the MT ring. In dividing cells, spindle MTs appeared as the chromosomes were condensed. During the early stages of expansion of the semicells, after cell division, the spindle MTs assumed a radial arrangement, moved, and settled in a position between the daughter chloroplasts. These MTs disappeared about 1.5 h after septum formation. As the new semicells were growing, wall MTs appeared, arranged transversely along the expanding wall. These transverse MTs disappeared gradually 4–5 h after septum formation, and only an MT ring remained near the boundary between the new and old semicells. The MT ring was present until the next cell division or expansion without cell division. During the latter course of development, transverse wall MTs were present only at the band-like expanding region. At the earlier stage of expansion without cell division, the short, hair-like MTs remained around the nucleus, but as time passed, both the hair-like MTs and, somewhat later, the transverse ones disappeared and only the MT rings remained. The remaining MT ring was not always positioned at the boundary between the expanding and the old cell region. The temporal relationships between the changes in MT arrangement, and the orientation and localization of cellulose-microfibril deposition are discussed.Abbreviations DAPI 46-diamino-2-phenylindole - EGTA ethyleneglycol-bis-(-aminoethylether)-N, N, N, N-tetraacetic acid - MT mierotubule - PMSF phenylmethylsulfonyl fruoride     

Calcium-mediated changes in peroxidase and O-diphenol oxidase activities of cotton fibres (Gossypium spp.) and its possible relationship to ABA

This work is an investigation of the influence of ABA and calcium on soluble and wall-bound activities of peroxidase and O-diphenol oxidase of cotton fibres at the stages of primary and secondary wall development, during incubation of intact fibres for 3h at 28°C. ABA (10 M) caused marked inhibition of enzyme activities in both the fractions, whereas calcium (1 mM) was promotory. The incorporation of 1 mM EGTA (a calcium chelator) and chlorpromazine (10 g cm^–3) (a calmodulin antagonist) resulted in decreased enzyme activities suggesting regulation of enzyme synthesis and/or secretion to the cell wall by calcium-calmodulin. As a general trend, the relative effect of Ca²⁺ on the activity of peroxidase in the wall was much greater than on the soluble activity, but this was not true of O-diphenol oxidase. It is inferred that ABA inhibits enzymic activities by inhibiting calmodulin synthesis or its mobilization to sites of action.Abbreviations ABA abscisic acid - EGTA ethylene glycol-bis( amino-ethyl ether)N,N tetraacetic acid - DAA days after anthesis     

Plasmid-mediated control of nodulation in Rhizobium trifolii

The nodulation ability was effectively eliminated from different Rhizobium trifolii strains incubated at elevated temperature (urkowski and Lorkiewicz, 1978). Non-nodulating (Nod^-) mutants were stable and no reversion of Nod^- to Nod⁺ phenotype was observed. Strains R. trifolii 24 and T12 which showed a high percentage of elimination of nodulation ability were examined in detail. Two plasmids were detected in strain 24 using neutral and alkaline sucrose gradient centrifugation of plasmid preparations. Molecular weights of the plasmids pWZ1 and pWZ2 were 460 Mdal and 190 Mdal, respectively. Rhizobium lysates labeled with ³H-thymidine and ultracentrifuged in caesium chloride — ethidium bromide gradients demonstrated a 40% reduction of the plasmid DNA content in R. trifolii 24 Nod^- mutants in comparison with the nodulating wild type strain 24. It was found further that non-nodulation of mutants 24 Nod^- was due to the absence of plasmid pWZ2. Sucrose gradient data also demonstrated that strain T12 contained two plasmids with molecular weights corresponding to those of pWZ1 and pWZ2, respectively. In Nod^- mutant clones derived from strain T12, pWZ2 plasmid was missing.Non Standard Abbreviations CCC covalently closed circular - OC open cirucular - Sarkosyl sodium N-lauroylsarcosinate     

Late type of daughter cell wall synthesis in one of the Chlorellaceae, <Emphasis Type="Italic">Parachlorella kessleri</Emphasis> (Chlorophyta,Trebouxiophyceae)

Autosporulation is a common mode of propagation for unicellular algae. Autospore-forming species of Chlorellaceae, Chlorella vulgaris Beijerinck, C. sorokiniana Shihira et Krauss, C. lobophora Andreyeva, and Parachlorella kessleri (Fott et Nováková) Krienitz et al. have glucosamine as the main constituent of their rigid cell wall. Recent phylogenetic analyses have showed that the Chlorellaceae divided into two sister groups: the Chlorella-clade and the Parachlorella-clade. We compared the cell wall structure and synthesis of the daughter cell wall in the four species by electron microscopy using rapid freezing and freeze substitution methods. The cell wall of C. vulgaris, C. sorokiniana, and C. lobophora consisted of an electron-dense thin layer with an average thickness of 17–20, 22, and 19 nm, respectively. In these three species, daughter cell wall synthesis occurred on the outer surface of the plasma membrane in the early cell-growth phase. The cell wall of P. kessleri, however, was electron-transparent and 54–59 nm in thickness. Ruthenium red staining of P. kessleri indicated that ruthenium-red-specific polysaccharides accumulated over the outer surface of the plasma membrane. Immunoelectron microscopic observation with an anti--1, 3-glucan antibody and staining with wheat germ agglutinin (WGA) indicated that the cell wall contained -1, 3-glucan and WGA specific N-acetyl--D-glucosamine. In P. kessleri, daughter cell wall synthesis began after successive protoplast division. The daughter cell wall synthesis during autosporulation in the four species of Chlorellaceae can be classified into two types—the early and the late types.     

Phase transitions in plant cuticles

The effect of temperature on wet plant cuticles has been investigated with the following techniques: Calorimetry, densitometry, spin-label electron-spin-resonance-(ESR)-spectroscopy, photo bleaching, and light and electron microscopy. At low temperatures cuticles ofCitrus aurantium L. andHedera helix show, at 16.3°C, a sharp transition (T0.5°C) with a latent heat of 4.7±0.5 J g^-1-cuticle. Below transition: The main orientation of the polymer matrix is parallel to the normal of the cuticle and the main orientation of the layer with soluble lipids is perpendicular to the normal. The cuticle is in a rigid state. Above transition (between 16.3°C and 38°C): Only the orientation of the polymer matrix has changed (tilted in parts). There exist several very sharp (T0.1°C) transitions (38°C, 41°C, 45°C, 49°C, ...) with a latent heat in the order of 0.4 J g^-1-cuticle. Above 38°C: The lamella of the soluble lipids is in a fluid state. Above 45°C there is a change in the molecular orientation of the soluble lipids as well as in the polymer matrix. The soluble lipids are mainly oriented parallel to the normal. The dry cuticles show no phase transition between 0°C and 200°C. At room temperature a dry/wet transition can be observed.Abbreviations ESR-spectroscopy electron-spin-resonance-spectroscopy