Thermodynamic analysis of nonelectrolyte sorption in plant cuticles: The effects of concentration and temperature on sorption of 4-nitrophenol

The sorption of 4-nitrophenol (4-NP) in enzymatically isolated cuticles ofLycopersicon esculentum fruits andFicus elastica leaves was studied as a function of temperature and solute concentration. Plots of the concentrations of 4-NP sorbed in the cuticle versus the equilibrium concentrations in the aqueous phase gave linear isotherms at low concentrations that tended to approach plateaus at higher sorbate concentrations ( 10 mmol·kg^-1). At low concentrations of sorbed 4-NP, cuticles have sorptive properties similar to those of organic solvents which are able to form intermolecular hydrogen bonds, while at higher concentrations their solid nature becomes apparent. During sorption of 4-NP the cutin matrix swells and new sorption sites are successively formed. The partition coefficients of 4-NP in the system cuticle/buffer are functions of temperature and concentration. At high sorbate concentrations (approx. 1 mol·kg^-1) they approach a value of 1. Different sorptive properties were observed for the cutin regions normally encrusted with soluble cuticular lipids (SCL) and those without SCL. Increasing temperature augmented the number of sorption sites in the cutin ofLycopersicon while no effect was observed withFicus. The changes of partial molar free energy (G ^o _tr), enthalpy (H ^o _tr), and entropy (S ^o _tr) for the phase transfer of 4-NP also depended on sorbate concentration: H ^o _tr and S ^o _tr were negative and steeply decreased at high sorbate concentrations. This is due to solute-solute interactions replacing solute-cutin interactions at high concentrations resulting in solid precipitates of solute within the cutin matrix. This formation of ordered solid domaines starting from a small number of nonelectrolyte molecules interacting with the cutin is proposed as a model for the intracuticular deposition of SCL.Abbreviations CM cuticular membrane - MX polymer matrix membrane - 4-NP 4-nitrophenol - SCL soluble cuticular lipids     

Enhancement of cadmium effects on growth and nutrient composition of birch (Betula pendula) by buthionine sulphoximine (BSO)

Binding of Cd to non-specific metal-binding peptides (phytochelatins)in birch roots has been suggested as an explanation for toleranceto Cd toxicity in birch (Betula pendula). In the present study,the tolerance of birch roots to Cd was further investigatedby using buthionine sulphoximine (BSO) as an inhibitor of phytochelatinsynthesis. Birch seedlings, grown in nutrient solution at pH4.2, were exposed to 0 or 2 µM CdCl₂ combined with 0 or0.1 mM BSO for 6 d. Plant growth (fresh weight increase andshoot to root dry weight ratio) and the nutrient compositionin fine roots, whole roots and shoots were determined. The effectsof Cd on growth confirms the results of earlier studies on birch,suggesting a reduced shoot growth, but preserved or stimulatedroot growth. When Cd and BSO were combined, overall plant growthwas severely reduced. BSO was also shown to aggravate Cd-inducedreductions of root and shoot concentrations of K, Ca and Mgbut to impede the accumulation of Cd. The results suggest that phytochelatins participate in protectingthe root against Cd interferences with growth, possibly by restrictingCd-induced changes in the nutrient composition of the plant. Key words: Betula pendula, buthionine sulphoximine, cadmium, phytochelatins, roots, tolerance     

The ultrastructure of the cuticle of Nematoda

 The ultrastructure of the body cuticle in species of six of seven representative genera of Stilbonematinae (Eubostrichus, Catanema, Laxus, Robbea, Leptonemella, and Stilbonema) was investigated using SEM and TEM techniques. Additionally, one species of Spirinia (Spiriniinae) and one of Desmodora (Desmodorinae) were studied for outgroup comparison. The body cuticle of all investigated stilbonematids shows a consistent pattern composed of specific elements in a characteristic arrangement to each other. This pattern does not occur in Stilbonematinae alone, but also in Desmodorinae and Spiriniinae. Furthermore, a comparison within the Desmodorida reveals that this cuticular pattern apparently is present in the cuticle of representatives of Monoposthiidae, Epsilonematidae, and Draconematidae. The present results lead to the following conclusions: (1) the cuticle of Stilbonematinae contains no autapomorphic characters for this taxon, (2) there is a common cuticular pattern within the Desmodorida, and (3) this pattern is an autapomorphic character for the order Desmodorida. Accepted: 4 February 1996     

Tn5 insertion mutants of Pseudomonas sp. 267 defective in siderophore production and their effect on clover (Trifolium pratense) nodulated with Rhizobium leguminosarum bv. trifolii

Pseudomonas sp. strain 267 isolated from soil promoted growth of different plants under field conditions and enhanced symbiotic nitrogen fixation in clover under gnotobiotic conditions. This strain produced pyoverdine-like compound under low-iron conditions and secreted vitamins of the B group. The role of fluorescent siderophore production in the beneficial effect of strain 267 on nodulated clover plants was investigated. Several non-fluorescent (Pvd^-) Tn5 insertion mutants of Pseudomonas sp. strain 267 were isolated and characterized. The presence of Tn5 insertions was confirmed by Southern analysis of EcoRI digested genomic DNA of each derivative strain. The siderophore-negative mutants were compared to the parental strain with respect to their growth promotion of nodulated clover infected with Rhizobium leguminosarum bv. trifolii 24.1. We found that all isolated Pvd^- mutants stimulated growth of nodulated clover plants in a similar manner to the parental strain. No consistent differences were observed between strain 267 and Pvd^- derivatives strains with respect to their plant growth promotion activity under gnotobiotic conditions.Dr Deryto died in august 1994     

Glutathione synthesis in maize genotypes with different sensitivities to chilling

The effect of chilling on enzymes, substrates and products of sulfate reduction, gultathione synthesis and metabolism was studied in shoots and roots of maize (Zea mays L.) genotypes with different chilling sensitivity. At full expansion of the second leaf, chilling at 12 °C inhibited dry weight increase in shoots and roots compared to controls at 25 °C and induced an increase in adenosine 5-phosphosulfate sulfotransferase and -glutamylcysteine synthetase (EC 6.3.2.2) activity in the second leaf of all genotypes tested. Glutathione synthetase (EC 6.3.2.3) activity was about one order of magnitude higher than -glutamylcysteine synthetase activity, but remained unchanged during chilling except for one genotype. During chilling, cysteine and glutathione content of second leaves increased to significantly higher levels in the two most chilling-tolerant genotypes. Comparing the most tolerant and most sensitive genotype showed that chilling induced a greater incorporation of³⁵S from [³⁵S]sulfate into cysteine and glutathione in the chilling-tolerant than in the sensitive genotype. Chilling decreased the amount of³⁵S-label incorporated into proteins in shoots of both genotypes, but had no effect on this incorporation in the roots. Glutathione reductase (EC 1.6.4.2) and nitrate reductase (EC 1.6.6.1) activity were constitutively higher in the chilling-tolerant genotypes, but showed no changes in most examined genotypes during 3 d at 12 °C. Our results indicate that in maize glutathione is involved in protection against chilling damage.Abbreviations APSSTase adenosine 5-phosphosulfate sulfotransferase - EC -glutamylcysteine - GR glutathione reductase - OSH glutathione - NR nitrate reductase We thank M. Suter for preparing [³⁵S]adenosine 5-phosphosulfate, Dr. A. Fleming (both our Institute) for correcting the English and M. Soldati (Eschlikon, Switzerland) for his help with the plant material. This work was supported by COST 814 Crop development for the wet and cool regions of Europe.     

In vitro Reconstitution of Epicuticular Wax Crystals: Formation of Tubular Aggregates by Long-Chain Secondary Alkanediols

Cuticles of several plant species are covered by tubular wax aggregates that are known to consist mainly of (S)-nonacosan-10-ol. The present work addresses the question whether minor wax components may additionally contribute to these tubules. Thin layer chromatography was used to prepare secondary alkanediol fractions from leaf cuticular waxes of Nelumbo nucifera and Thalictrum flavum, containing nonacosane-3,10-diol, nonacosane-4,10-diol, nonacosane-5,10-diol, nonacosane-7,10-diol, nonacosane-9,10-diol and nonacosane-10,13-diol. From organic solutions all these compounds crystallized in tubular shapes. Possible crystal structures of relevant alkanediol isomers are proposed, in analogy to the lattice geometries of comparable aliphatic compounds. The resulting structural model shows that nonacosan-10-ol and various secondary alkanediols may join in metastable mixed crystals. According to the structural model proposed the admixture of alkanediols to nonacosan-10-ol aggregates should enhance the stability of their tubular habit.     

Evaluation of the palatability of chrysomelid larvae containing anthraquinones to birds

Chrysomelid larvae of the subfamily Galerucinae, tribe Galerucini, are known to contain 1,8-dihydroxylated 9,10-anthraquinones. Since nonhydroxylated 9,10-anthraquinone is the active agent in several commercial products sold to protect seeds against birds, we suggested that the naturally occurring dihydroxylated anthraquinones of galerucine larvae may also act as protective devices against bird predation. Tits (Parus spp.) are potential predators of larvae of the tansy leaf beetle, Galeruca tanaceti, and the elm leaf beetle, Xanthogaleruca luteola. To investigate the palatability of these chrysomelid larvae to birds, we offered them with mealworms and Calliphora pupae, respectively, as controls in dual choice bioassays to eight singly kept, naive tits (five P. major and three P. ater individuals). The bioassays were limited to 5 days, during which larvae were offered daily for 2 h (X. luteola) and 3 h (G. tanaceti), respectively. Every day, the birds significantly avoided uptake of G. tanaceti and X. luteola. More than 98% of the control food was consumed daily, whereas the percentage of chrysomelid larvae totally eaten never surpassed 6.6% for G. tanaceti and 51.8% for X. luteola. In order to determine whether this avoidance was due to the anthraquinones of the chrysomelid larvae, mealworms and Calliphora pupae, respectively, were treated with these compounds in concentrations equivalent to the natural ones. Dual choice bioassays with treated and untreated prey were conducted, again for 5 days with a daily 2- or 3-h test period, respectively. The tits ate all or nearly all treated and untreated food items every day. However, during the 5-day test period the tits learnt to take up the control insects significantly earlier than the treated ones; the food containing anthraquinones was not consumed as readily as the control, which suggest aversive learning based on distastefulness. The efficiency of anthraquinones in protecting galerucine larvae against bird predation is discussed with special respect to learning behavior and factors which might delay or mask learning of avoidance.     

Photoinhibition and light-dependent turnover of the D1 reaction-centre polypeptide of photosystem II are enhanced by mineral-stress conditions

The function of photosystem II (PSII) and the turnover of its D1 reaction-center protein were studied in spinach (Spinacia oleracea L.) plants set under mineral stress. The mineral deficiencies were induced either by supplying the plants with an acidic nutrient solution or by strongly reducing the supply of magnesium alone or together with sulfur. After exposure for 8–10 weeks to the different media, the plants were characterized by a loss of chlorophyll and an increase in starch content, indicating a disturbance in the allocation of assimilates. Depending on the severity of the mineral deficiencies the plants lost their ability to adapt even to moderate iradiances of 400 mol photons·m^–2·s^–1 and became photoinhibited, as indicated by the decrease in F_v/F_m (the ratio of yield of variable fluorescence to yield of maximal fluorescence when all reaction centers are closed). The loss of PSII function was induced by changes on the acceptor side of PSII. Fast fluorescence decay showed a loss of PSII centers with bound Q_B, the secondary quinone acceptor of PSII, and a fast reoxidation kinetic of q _a ^- , the primary quinone acceptor of PSII, in the photoinactivated plants. No appreciable change could be observed in the amount of PSII centers with unbound Q_B and in Q_B-nonreducing PSII centers. Immunological studies showed that the contents of the D1 and D2 proteins of the PSII reaction center and of the 33-kDa protein of the water-splitting complex were diminished in the photoinhibited plants, and the occurrance of a new polypetide of 14 kDa that reacted with an antibody against the C-termius of the D1 protein. As shown by pulse-labelling experiments with [¹⁴C]leucine both degradation and synthesis of the D1 protein were enhanced in the mineral-deficient plants when compared to non-deficient plants. A stimulation of D1-protein turnover was also observed in pH 3-grown plants, which were not inhibited at growth-light conditions. Obviously, stimulation of D1-protein turnover prevented photoinhibition in these plants. However, in the Mg- and Mg/S-deficient plants even a further stimulation of D1-protein turnover could not counteract the increased rate of photoinactivation.Abbreviations amp_(f,m,s) amplitude of the fast, (medium and slow) exponential component of fluorescence decay - F_m yield of maximum fluorescenc when all reaction centers are closed - F_o yield of intrinsic fluorescence at open PSII reaction centers in the dark - F_v yield of variable fluorescence, (difference between F_m and F_o) - LHC light-harvesting complex - PFD photon flux density - Q_A primary quinone acceptor of PSII - Q_B secondary quinone acceptor of PSII Dedicated to Professor Dr. Dres. hc. Achim Trebst on the occasion of his 65th birthdayThis work was supported by grants from the BMFT and the Ministerium für Umwelt, Raumordnung and Landwirtschaft, Nordrhein-Westfalen. The authors thank H. Wietoska and M. Bronzel for skilful technical assistance.     

Definition and characterization of an artificial En/Spm-based transposon tagging system in transgenic tobacco

A transposon tagging system for heterologous hosts, based on the maize En/Spm transposable element, was developed in transgenic tobacco. In this system, the two En-encoded trans-acting factors necessary for excision are expressed by fusing their cDNAs to the CaMV 35S promoter. The dSpm receptor component is inserted in the 5-untranslated leader of the bar gene. Germinal revertants can therefore be selected by seed germination on L-PPT-containing medium or by spraying seedlings with the herbicide Basta. Using this bar-based excision reporter construct, an average frequency of germinal excision of 10.1% was estimated for dSpm-S, an En/Spm native internal deletion derivative. Insertion of En-foreign sequences in a receptor, such as a DHFR selectable marker gene in dSpm-DHFR, does not abolish its capacity to transpose. However, dSpm-DHFR has a lower frequency of somatic and germinal excision than dSpm-S. Revertants carrying a transposed dSpm-DHFR element can be selected with methotrexate. Germinal excision is frequently associated with reinsertion but, as in maize, dSpm has a tendency to integrate at chromosomal locations linked to the donor site. Concerning the timing of excision, independent germinal transpositions are often found within a single seed capsule. All activity parameters analysed suggest that transposon tagging with this system in heterologous hosts should be feasible.     

The Casparian Strip of Clivia miniata Reg. Roots: Isolation,Fine Structure and Chemical Nature*

The root endodermis of Clivia miniata Reg. was successfully isolated using the cell wall degrading enzymes cellulase and pectinase. The enzymes did not depolymerize those regions of the primary cell walls of anticlinal endodermal root cells where the Casparian strips were located. Since the endodermis of C. miniata roots remained in its primary developmental state over the whole root length, endodermal isolates essentially represented Casparian strips. Thus, sufficient amounts of isolated Casparian strips could be obtained to allow further detailed investigations of the isolates by microscopic, histochemical and analytical methods. Scanning electron microscopy revealed the reticular structure of the Casparian strips completely surrounding the central cylinder of the roots. Whereas in younger parts of the root only the anticlinal cell walls of the endodermis remained intact in the isolates, in older parts of the root the periclinal walls also restricted enzymatic degradation due to the deposition of lignin. Extracts of the isolates with organic solvents did not reveal any wax-like substances which might have been deposited within the cell wall forming a transport barrier, as is the case with cutin and suberin. However, several histochemical and analytical methods (elemental analysis and FTIR spectroscopy) showed that the chemical nature of the Casparian strips of C. miniata roots can definitely be a lignified cell wall. These findings are in complete agreement with studies carried out at the beginning of this century on the chemical nature of the Casparian strips of several other plant species. The implications of these results concerning apoplasmatic transport of solutes and water across Casparian strips are discussed.