Utilization of glycine and serine as nitrogen sources in the roots of Zea mays and Chamaegigas intrepidus

Glycine and serine are potential sources of nitrogen for the aquatic resurrection plant Chamaegigas intrepidus Dinter in the rock pools that provide its natural habitat. The pathways by which these amino acids might be utilized were investigated by incubating C. intrepidus roots and maize (Zea mays) root tips with [(15)N]glycine, [(15)N]serine and [2-(13)C]glycine. The metabolic fate of the label was followed using in vivo NMR spectroscopy, and the results were consistent with the involvement of the glycine decarboxylase complex (GDC) and serine hydroxymethyltransferase (SHMT) in the utilization of glycine. In contrast, the labelling patterns provided no evidence for the involvement of serine:glyoxylate aminotransferase in the metabolism of glycine by the root tissues. The key observations were: (i) the release of [(15)N]ammonium during [(15)N]-labelling experiments; and (ii) the detection of a characteristic set of serine isotopomers in the [2-(13)C]glycine experiments. The effects of aminoacetonitrile, amino-oxyacetate, and isonicotinic acid hydrazide, all of which inhibit GDC and SHMT to some extent, and of methionine sulphoximine, which inhibited the reassimilation of the ammonium, supported the conclusion that GDC and SHMT were essential for the metabolism of glycine. C. intrepidus was observed to metabolize serine more readily than the maize root tips and this may be an adaptation to its nitrogen-deficient habitat. Overall, the results support the emerging view that GDC is an essential component of glycine catabolism in non-photosynthetic tissues.     

Viability studies on actinomycetes

Eighty-nine Actinomycetes strains were tested for their viability, morphological and physiological characteristics after being kept under paraffin oil overlay and distilled water for a period between 10–30 years. Most of the studied strains belong to the “Lorenzo De Montemayor” collection. Almost all the recovered strains were 28–30 years old and had never been subcultured since the paraffin oil was overlaid. 71.4% of viable Streptomycetes strains had been kept on Sabouraud-dextrose agar and 28.6% were kept on Negroni and Bonfiglioli-medium. Streptomyces violaceusruber produced its characteristic pigment even after 28 years under these conditions. All of the recovered strains were tested for their biological activity, but only Streptomyces lavendulae showed growth-inhibition against Staphylococcus aureus and Bacillus subtilis. This revised version was published online in June 2006 with corrections to the Cover Date.     

Water uptake by roots of maize and sunflower affects the radial transport of abscisic acid and its concentration in the xylem

The radial movement of cis-abscisic acid (ABA) has been investigated in young excised roots of Zea mays L. and Helianthus annuus L. which were grown hydroponically. In addition to the symplastic path, ABA was largely translocated across the root apoplast by solvent drag with the water in the transpiration stream. On the apoplastic path ABA may even cross the endodermis. Depending on the ABA concentration of the medium (range: 5–500 nM) and in the root apoplast, the solvent-drag component of the flow of ABA counteracted the dilution of ABA in the xylem caused by transpirational water flow. Acidification of the rhizosphere and of the root apoplast increased the apoplastic transport component. In sunflower, the apoplastic flow of ABA was significantly weaker than in maize roots. This was also indicated by the larger apparent reflection coefficient (σ_ABA) of sunflower roots for ABA (sunflower: σ_ABA = 0.97 ± 0.02, n = 6 roots; maize: σ_ABA = 0.68 ± 0.06, n = 6 roots; ±SD). For both species, σ_ABA was smaller than unity. Root reflection coefficients were affected by factors such as pH, ABA concentration of the medium, and by the suction force applied to excised root systems. Due to the complex composite structure of the permeation barrier in the root, the reflection coefficient estimated from solvent drag is also complex. Since unstirred layers affected the absolute value of the reflection coefficient, σ_ABA has been termed `apparent'. It is concluded that the pH and ABA concentration of the soil solution as well as the transpiration rate (suction force) modify the intensity of the root-to-shoot signal which is influenced by an apoplastic bypass flow of ABA. The latter may be substantially affected by the existence of Casparian bands in the exodermis, which were lacking in the roots studied in this paper. Received: 25 February 1998 / Accepted: 16 July 1998     

Highly specific PCR-diagnosis to determine Pseudomonas solanacearum strains of different geographical origins

 Using a PCR-based assay with highly specific primers, we were able to clearly identify all of 28 different Pseudomonas solanacearum strains, whereas none of the other bacteria tested gave a cross reaction. The PCR sensitivity in standard dilution experiments of pure strains was in the range of 10 to 100 cells. The assay was also investigated for its suitability in routine diagnosis of potato tubers and tomato plants inoculated with various amounts of P. solanacearum; it reached a sensitivity of 10³ cells per specimen. The region between primers PS96H and PS96I was sequenced for the first time and aligned. A total of 17 P. solanacearum strains have been sequenced, resulting in six different sequence groups. When the variable sequence was analyzed, a high correlation between point mutations and geographical origin of the P. solanacearum strains was revealed. The PCR assay described in this study combined with automatical sequencing of the amplificated region provides a powerful tool for the epidemiology of P. solanacearum. Received: 1 September 1997 / Accepted: 15 October 1997     

Comparison of Photosynthetic Responses of the Sympatric Tropical C3 Species Clusia multiflora H. B. K. and the C3-CAM Intermediate Species Clusia minor L. to Irradiance and Drought Stress in a Phytotron1

Abstract: Clusia multiflora H. B. K., an obligate C₃ species and Clusia minor L. a C₃/CAM intermediate species, are two physio-types of a similar morphotype. They can sympatrically occupy secondary savanna sites exposed to high insolation in the tropics. In C. multiflora severe stress, i.e., switching shade-grown plants to high light plus drought, resulted in leaves browning or yellowing and becoming necrotic. However, in long-term light stress C. multiflora was able to grow new leaves with their photosynthetic apparatus fit for high light conditions. Shade-grown C. minor readily overcame switching to high light conditions and drought, responding by a rapid change from C₃ photosynthesis to CAM. Decreasing _soil led to increased abscisic acid levels in the leaves of C. minor, however CAM induction was not directly related to this and was mainly determined by increased PPFD. Both species were capable of rapid accumulation of zea-xanthin for acute photoprotection following high PPFD exposure. The maximum capacity for zeaxanthin accumulation was larger in C. minor, but under steady high PPFD it only partially made use of this capacity, relying on high internal CO₂ concentrations of Phase Ill of CAM, in addition to zeaxanthin, for acute photo-protection. Thus, by different means the two species perform well under high light conditions. However, C. multiflora needs time for development of adapted leaves under such stress conditions while the more flexible C. minor can readily switch from low light to high light conditions.     

Cellular Fatty Acid Composition of Nine Pathovars of Xanthomonas campestris

Cellular fatty acids of 80 strains of Xanthomonas campestris, representing 9 different pathovars, were analyzed by gas-liquid chromatography and mass spectrometry. A total of 48 fatty acids were identified, the most important being the 16:0 (averaging at least 4.5 % of the total), the cis- and trans- 9 16 : 1 (over 14.4 %), and the iso and anteiso 15 : 0 (over 30 %). Other major fatty acids (averaging over 1 % of total) were the saturated 14 : 0 and 15 : 0, the hydroxy-substituted iso 3-OH 11 : 0, 3-OH 12 : 0 and iso 3-OH 13 : 0, and the branch-chained iso 11 : 0, iso 16 : 0, iso 17 : 1, iso 17 : 0 and anteiso 17 : 0. Of 33 minor fatty acids detected and identified, only 7 have been previously reported in the xanthomonads. Significant differences in mean percentages of 5 major fatty acids and 4 (chemical) class totals were detected among pathovars, which statistically segregated into three groups by rank analysis. X. campestris pv. dieffenbachiae was in a group by itself; pvs. campestris, citri (pathotypes A and B), manihotis, phaseoli, pruni and vesicatoria were in a seond group, and pvs. glycines, begonia and citri (pathotype E) were in a third.     

Uptake of jasmonic acid and related compounds by mesophyll protoplasts of the barley leaf

Jasmonic acid (JA) permeates the plasma membrane of mesophyll cells by diffusion as the lipophilic undissociated JAH molecule probably without the participation of a saturable uptake component. The mesophyll plasma membrane is nearly impermeable to the JA anion. The permeability coefficients of JA and several JA derivatives (its methyl ester (JAMe), 7-iso-cucurbic acid (7-iso-CA), 6-epi-7-iso-cucurbic acid (6-epi-7-iso-CA), and both stereoisomers of the JA leucine conjugate ((+)-JA-Leu and (-)-JA-Leu)) were determined and used in a simplified mathematical model to predict stressdependent JA redistribution between cytosol and apoplast in comparison with ABA. The redistribution of JA takes place similar to ABA; however, its velocity is much higher because of the high JA membrane permeability. When the permeability coefficients for the mesophyll plasma membrane are plotted double-logarithmically against the ratio of the distribution coefficient and the molecular ratio to the power of 1.5 (K_DM_r ^–1.5), two straight lines result for two different classes of compounds. The permeability coefficients of JA conjugates are lower than that of the free acid by approximately one order of magnitude, but they are still significantly higher than that of ABA.     

Nonhomologous DNA end joining of synthetic hairpin substrates in Xenopus laevis egg extracts.

Processes of DNA end joining are assumed to play a major role in the elimination of DNA double-strand breaks (DSB) in higher eucaryotic cells. Linear plasmid molecules terminated by nonhomologous restriction ends are the typical substrates used in the analysis of joining mechanisms. However, due to their limited structural variability, DSB ends generated by restriction cleavage cover probably only part of the total spectrum of naturally occurring DSB termini. We therefore devised novel DNA substrates consisting of synthetic hairpin-shaped oligonucleotides which permit the construction of blunt ends and 5'- or 3'-protruding single-strands (PSS) of arbitrary sequence and length. These substrates were tested in extracts of Xenopus laevis eggs known to efficiently join linear plasmids bearing nonhomologous restriction termini (Pfeiffer and Vielmetter, 1988). Sequences of hairpin junctions indicate that the short hairpins are joined by the same mechanisms as the plasmid substrates. However, the bimolecular DNA end joining reaction was only detectable when both hairpin partners had a minimal duplex stem length of 27bp and their PSS-tails did not exceed 10nt.     

Evaluation of an automated fluorescent antibody procedure for detection of Salmonella in foods and feeds.

A prototype automated system using fluorescent antibody (FA) was evaluated for rapid detection of salmonellae in foods. Samples were enriched in selenite cystine and tetrathionate broths. After incubation, both were transferred into fresh selenite cystine for a 4-h "post-enrichment" to dilute possible background fluorescence from product. These cultures were then analyzed automatically, and results were compared with those obtained by the methods of the Association of Official Analytical Chemists (AOAC). Initially, 167 samples of milk powder, dried yeast, and imported frog legs were examined. The AOAC and automated FA methods correlated well with all samples but frog legs. Difficulty with the latter was caused by procedural and mechanical problems coupled with high numbers of competing microorganisms in post-enrichment cultures. Modification of procedure and partial redesign of equipment corrected these difficulties, and excellent correlation was obtained with another 116 frog leg samples. All 89 AOAC-confirmed positives were also detected by the automated FA method, and there were only 4% false FA positives. The system shows potential for screening products for salmonellae; however, all positives should be confirmed by manual biochemical and serological methods.     

Compartmental distribution and redistribution of abscisic acid in intact leaves

Using a computer model written for whole leaves (Slovik et al. 1992, Planta 187, 14–25) we present in this paper calculations of abscisic acid (ABA) redistribution among different leaf tissues and their compartments in relation to stomatal regulation under drought stress. The model calculations are based on experimental data and biophysical laws. They yield the following results and postulates: (i) Under stress, compartmental pH-shifts come about as a consequence of the inhibition of the pH component of proton-motive forces at the plasmalemma. There is a decrease of net proton fluxes by about 8.6 nmol · s^–1 · m^–2. (ii) Using stress-induced pH-shifts we demonstrate how stress intensities can be quantified on a molecular basis. (iii) As the weak acid ABA is the only phytohormone which behaves in vivo and in vitro ideally according to the Henderson-Hasselbalch equation, pH-shifts induce a complicated redistribution amongst compartments in the model leaf. (iv) The final accumulation of ABA in guard-cell walls is intensive: up to 16.1-fold compared with only up to 3.4-fold in the guard-cell cytosol. We propose that the binding site of the guard-cell ABA receptor faces the apoplasm. (v) A twoto three-fold ABA accumulation in guard-cell walls is sufficient to induce closure of stomata. (vi) The minimum time lag until stomata start to close is 1–5 min; it depends on the stress intensity and on the guard-cell sensitivity to ABA: the more moderate the stress is, the later stomata start to close or they do not close at all. (vii) In the short term, there is almost no influence of the velocity of pH-shifts on the velocity of the ABA redistribution, (viii) Six hours after the termination of stress there is still an ABA concentration 1.4-fold the initial level in the guard-cell cytosol (delay of ABA relaxation, aftereffect), (ix) The observed induction of net ABA synthesis after onset of stress may be explained by a decrease in cytosolic ABA degradation. About 1 h after onset of stress the model leaf would start to synthesise ABA (and its conjugates) automatically, (x) This ABA net synthesis serves to inform roots via an increased ABA concentration in the phloem sap. The stress-induced ABA redistribution is per se not sufficient to feed the ploem sap with ABA. (xi) The primary target membrane of stress is the plasmalemma, not thylakoids. (xii) The effective stress sensor, which induces the proposed signal chain finally leading to stomatal closure, is located in epidermal cells. Mesophyll cells are not capable of creating a significant ABA signal to guard cells if the epidermal plasmalemma conductance to undissociated molecular species of ABA (HABA) is indeed higher than the plasmalemma conductance of the mesophyll (plasmodesmata open), (xiii) All model conclusions which can be compared with independent experimental data quantitatively fit to them. We conclude that the basic experimental data of the model are consistent. A stress-induced ABA redistribution in the leaf lamina elicits stomatal closure.Abbreviations ABA abscisic acid - CON vacuolar ABA conjugates We are grateful to Prof. U. Heber (Lehrstuhl Botanik I, University of Würzburg, FRG) for stimulating discussions. This work has been performed within the research program of the Sonderforschungsbereich 251 (TP 3 and 4) of the University of Würzburg. It has been also supported by the Fonds der Chemischen Industrie.