Chloroplasts and the biosynthesis and catabolism of abscisic acid

Chloroplast preparations from the mesocarp ofPersea gratissima and from light-grown shoots ofPisum sativum were unable to synthesize abscisic acid (ABA) from mevalonolactone, mevalonic acid, or isopentenyl pyrophosphate. Similar plastid preparations transformed [2-¹⁴C]ABA into acidic products that were chromatographically similar to those generated byP. gratissima mesocarp slices and excised shoots ofP. sativum. Attempts to increase ABA catabolism in chloroplast preparations using sedimentation through Percoll to remove associated proteases also reduced the capacity for ABA catabolism, suggesting that such catabolism arose from contaminating, cytoplasmic enzymes. Both lincomycin and chloramphenicol inhibited the catabolism of ABA by excised shoots ofP. sativum but had little effect on either ABA biosynthesis or ABA catabolism in mesocarp fromP. gratissima. These processes were inhibited markedly by cycloheximide.     

A macerating ammonium oxalate fixative and schedule for some root meristems

0.25 % ammonium oxalate in a mixture of ethyl alcohol, chloroform, formaldehyde, acetic acid and hydrochloric acid works as a very fast and efficient macerating fixative for the root meristems ofAllium cepa, Allium sativum, Ornithogalum thyrsoides, Pisum sativum andVicia faba. Slicing the tip region of excised meristems hastens spindle poison pretreatment, fixation and staining. Owing to the dissolution of the middle lamella and eliminating a physical barrier to permeability an extremely rapid fixation and staining schedule involving several stains has thus been developed.     

Antigenic properties of T cell antigen-specific receptors isolated from the surface of rabbit and mouse spleen and lymph node cells

The presence ofa allotypic determinants was tested in fractions obtained by gel filtration of antigen-specific receptors isolated by immunoadsorption from lymphoid cells of antigen-stimulateda3-3 rabbits. This technique, as well as the inhibition of the reaction of isolated receptors with anti-T cell receptor antisera (anti R) by anti-a3 antibodies failed to demonstrate the presence of a allotypie determinants. The inhibitory effect of antigen-specific receptors isolated from the lymphoid cells of stimulated A/J mice on the cytotoxic effect of anti-Ia antibodies on mouse spleen cells in the presence of rabbit complement was tested. All preparations inhibited the cytotoxic reaction with the average effectivity of 60%. In order to confirm the presence of Ia determinants on the rabbit and mouse T cell receptor molecules it was shown that the reactions of three anti-R antisera with 12 different receptor preparations were inhibited by anti-la antibodies. SDS-PAGE analyses of¹²⁵I-labelled mouse specific receptors and the precipitate obtained by anti-R antisera showed that T cell receptors were present in fractions with molar mass 100 and 85 kg/mol. The molar mass of the former fraction after reduction and alkylation was 45 kg/mol.     

Conversion of 1′-deoxy-2H-ABA to2H-ABA in cell-free extracts fromCercospora rosicola

Abscisic acid (ABA) biosynthesis in the fungusCercospora rosicola has been studied previously by feeding potential intermediates to growing cultures and examining the products. The present work describes the isolation and partial characterization of an enzyme system which catalyzes the last step in this pathway, the conversion of 1′-deoxy-²H-ABA to²H-ABA. Enzyme extracts were prepared from cold-pressed mycelia of actively growingC. rosicola in tricine buffer containing sucrose and β-mercaptoethanol. Low-speed supernatants and resuspended microsomal preparations were active in converting 1′-deoxy-²H-ABA to²H-ABA. Optimum conditions for the reactions were established at 10 μM substrate, 300 μl enzyme extract in a total volume of 1 ml. Reaction products were chromatographed by reverse-phase high-performance liquid chromatography (HPLC). The presumptive ABA fractions were collected and the²H-ABA was measured by combined gas chromatography-mass spectrometry (GC-MS) with an²H-ABA standard. Most of the enzyme activity was found in the microsomal fraction. Typical reaction rates were on the order of 1.5 pmol²H-ABA formed/min/mg protein. The reaction required NADPH and was enhanced by FAD. Triarimol, a substituted pyrimidine, which inhibits ABA synthesis inC. rosicola, had no effect on the conversion of 1′-deoxy-²H-ABA to²H-ABA.     

A 1,4-β-D-glucan-synthase system fromDictyostelium discoideum

Particulate membrane preparations have been isolated from culminatingDictyostelium discoideum cells. The preparations incorporated glucose from uridine 5′-diphosphate-glucose into a glucose polymer or polymers. These have been shown to be homopolymers ofβ-linked glucose. A high percentage (78% by methylation analysis) of the linkages formed are 1,4-linkages and a lower percentage (12%) are 1,3-linkages. The glucan-synthase complex present in the particulate membrane preparation has an apparent K_m of 0.28 mM and a V_max of 1.59 nmol·min^?1·(mg protein)^?1. The enzyme system is dependent upon Mg²⁺ and cellobiose for maximal activity, but is inhibited by millimolar levels of Ca²⁺. Particulate membrane preparations were made from cells at various times during a synchronous developmental time course and demonstrated that the glucan-synthase activity appeared at the tight-aggregate stage of development.     

Growth correlations inBryophyllum leaves and exogenous growth regulators

Growth correlations in leaves ofBryophyllum may be recognized by the development of marginal shoots varying in their particular lamina regions, the correlative inhibition increasing from the top to the base. Cytokinins extend their promoting action inB. crenatum leaves farther in the apical than in the basal direction. In the uppermost leaves ofB. daigremontianum they evoke the development of marginal shoots with flowers in the apical and of vegetative shoots in the basal region. Less suitable for this research are auxins, gibberellins, and triiodobenzoic acid which are unable to stimulate the development of marginal shoots on leaves grown out under short days if not supplemented by cytokinins. Only TIBA induces under long days formation of adventitious shoots directly from the lamina surface on leaves developed below the terminal ring fasciation exhibiting at free ends of connate leaves normal marginal shoots. Under short days a complete reduction of lateral teeth takes place inB. verticillatum both on the extremity of ring fasciation and on the uppermost leaves, the marginal shoots being formed under the subsequent long days only in the angles between the ring fasciation parts. Furthermore the correlative inhibition of the leaf causes anisophylly in marginal shoots in the same way as that of axillaries inBryophyllum.     

High-Frequency Regeneration by Abscisic Acid (ABA) from Petiole Callus of Jatropha curcas

Jatropha curcas L. is attaining worldwide interest as an important biofuel crop. Experiments were conducted to improve the prevailing micropropagation technique as well as to develop a new ex vitro rooting method for J. curcas plant regeneration. Regeneration and ex vitro rooting efficiency was enhanced by augmenting the culture medium with abscisic acid (ABA). Different concentrations of 6-benzylaminopurine (BAP) and indole-3-butyric acid (IBA) were tested for callus generation from both in vitro and in vivo explants (leaf and petiole) on Murashige and Skoog (MS) medium. The best regenerative callus was achieved on MS medium supplemented with BAP (4.44 μM) and IBA (2.45 μM) from in vitro-cultured petioles. Highest regeneration (91%) was achieved by culturing petiole callus on MS medium supplemented with BAP (8.88 μM), IBA (0.49 μM), and ABA (1.9 μM), whereas 61% regeneration was obtained from in vitro leaf callus. Shoot proliferation and elongation was achieved on BAP (2.22 μM) and IAA (8.56 μM) with 10–13 shoots per explants. Highest rooting (65%) was achieved from M1 shoots (BAP, IAA, and ABA) on MS medium supplemented with IBA (2.45 μM), naphthaleneacetic acid NAA (0.54 μM), and 0.02% activated charcoal. Ex vitro rooting of 1-mo-old M1 shoots obtained from the charcoal-containing medium resulted optimum rooting (>72%) when transferred to polybags containing sterile sand. The plantlets were successfully acclimatized in soil with more than 98% survival rate in the greenhouse.     

Amino acid conjugates as metabolites of the plant growth regulator dihydrojasmonic acid in barley (Hordeum vulgare)

The biotransformation of [2-¹⁴C](±)9, 10-dihydrojasmonic acid (DJA) was studied in excised shoots of 6-day-old barley seedlings after 72 h. From the ethyl acetate extract, some minor metabolites were isolated and purified by DEAE-Sephadex A-25 chromatography, thin-layer chromatography (TLC), C₁₈-cartridges, and high-performance liquid chromatography (HPLC). The structural identification of these metabolites was performed by gas chromatography-mass spectrometry (GC-MS), circular dichroism (CD), and amino acid analysis, and the following amino acid conjugates were found:N-[(?)9,10-dihydrojasmonoyl]valine,N-[(?)9,10-dihydrojasmonoyl]isoleucine,N-[9,10-dihydrojasmonoyl]leucine,N-[11-hydroxy-9,10-dihydrojasmonoyl]valine,N-[11-hydroxy-9,10-dihydrojasmonoyl]isoleucine,N-[12-hydroxy-9,10-dihydrojasmonoyl]isoleucine; and the cucurbic acid-related compoundsN-{[3-hydroxy-2(4-hydroxypentyl)-cyclopent-1-yl]-acetyl}isoleucine andN-{[3-hydroxy-2(5-hydroxypentyl)-cyclopent-1-yl]-acetyl}isoleucine. The results suggest conjugation with isoleucine and valine, as well as preferential hydroxylation at position C-11 or hydrogenation at position C-6, as being important steps in the metabolism of (±)DJA in barley shoots.     

The Effect of Nutritional Factors and Plant Growth Regulators on Micropropagation and Production of Phenolic Acids and Saponins from Plantlets and Adventitious Root Cultures of Eryngium maritimum L.

Eryngium maritimum L. is a valuable medicinal species, but since it is protected plant, collection from natural populations is forbidden. Therefore, establishing an efficient system for micropropagation of this species is desirable. To determine the optimal nutritional factors needed for shoot multiplication, root development and secondary metabolites accumulation, different media and plant growth regulators were tested. The highest plant regeneration efficiency (over 96 %), with 4.4 shoots per explant was induced on Murashige and Skoog (MS) medium supplemented with 1.0 mg L^?1 benzyladenine (BA) and 0.1 mg L^?1 indole-3-acetic acid (IAA). The in vitro-regenerated shoots were rooted (83.3–100 %) and transferred to an experimental plot with 62 % efficiency. Flow cytometric analysis revealed no variation in nuclear DNA content in field- and in vitro-delivered plant material. Ultra high performance liquid chromatography (UHPLC) indicated that multiple shoots and roots from in vitro-regenerated plantlets and adventitious root cultures maintained the production of rosmarinic (RA) and chlorogenic (CGA) acids and triterpenoid saponins found in the rosette leaves and roots of E. maritimum intact plants. UHPLC revealed a 12-fold increase of RA and CGA and 3.2-fold higher accumulation of triterpenoid saponins in roots of in vitro-derived plantlets in comparison to roots from field-grown plants. Adventitious root cultures allowed continuous growth of excised root in liquid media with or without exogenous auxins. The roots grown in liquid medium supplemented with 0.1 mg L^?1 IAA showed higher (227-fold) phenolic acids accumulation than those without auxin. Obtained results confirmed that micropropagation is a useful strategy in the protection of endangered species and a renewable source of a high quality plant material for secondary metabolites production.     

Effect of benzylaminopurine and thidiazuron onin vitro shoot proliferation ofTilia cordata MILL.,Sorbus aucuparia L. andRobinia pseudoacacia L.

Benzylaminopurine and thidiazuron stimulated shoot proliferation ofTilia, Sorbus andRobinia. Low concentration of BAP (0.2—1.0 mg I^?1) promoted axillary bud formation and shoot elongation. Thidiazuron displayed high cytokinin activity at very low concentrations (0.002—0.05 mg I^?1). Shoot number induced on media containing thidiazuron was large. Numerous shoots were produced on the media containing BAP together with thidiazuron. Shoots produced on media containing thidiazuron or BAP together with thidiazuron rooted after transfer to medium supplemented with low concentration of auxin (IBA or NAA).     

Shoot regeneration from mesophyll protoplasts and leaf explants of Rehmannia glutinosa

Mesophyll protoplasts obtained from leaves of shoot cultures of Rehmannia glutinosa were cultured in Murashige and Skoog (1962) liquid or liquid-over-agar medium containing 2.0 mg L^?1 naphthaleneacetic acid and 0.5 mg L^?1 benzylamino purine. An amino acid mixture of glutamine, arginine, glycine, and aspartic acid promoted sustained protoplast division, with an average plating efficiency of 27%. Protoplast-derived colonies formed callus which readily regenerated shoots on fransfer to Murashige and Skoog based agar medium with 2.0 mg L^?1 indoleacetic acid and 1.0 mg L^?1 benzylamino purine. Leaf explants also showed a marked capacity for shoot regeneration in culture.     

Regulation of citrate synthase activity ofSaccharomyces cerevisiae

Citrate synthase activity ofSaccharomyces cerevisiae was determined by a radioactive assay procedure and the reaction product,¹⁴C-citric acid, was identified by chromatographic techniques. ATP, d-ATP, GTP and NADPH were most inhibitory to the citrate synthasein vitro. The activity was inhibited to a lesser extent by ADP, UTP, and NADP whereas, AMP and CTP were much less inhibitory. NADH, like NAD, glutamic acid, glutamine, arginine, ornithine, proline, aspartic acid and α-ketoglutarate exhibited no inhibition. These results have been discussed in the light of the role of citrate synthase for the energy metabolism and glutamic acid biosynthesis.     

Pseudomonas aeruginosa contains an inducible methylcitrate synthase

Assaying for enzymes of propionyl-CoA catabolism in crude extracts ofPseudomonas aeruginosa was complicated by the presence of a short-chain acyl-CoA hydrolase. Interference by the hydrolase was avoided by using permeabilized whole cells. Cells treated this way exhibited low or undetectable levels of propionyl-CoA carboxylase, propionyl-CoA dehydrogenase, and α-hydroxyglutarate synthase, enzymes initiating propionyl-CoA catabolism in many prokaryotes, but did contain high levels of methylcitrate synthase, an enzyme of the methylcitrate cycle for propionyl-CoA catabolism. Gel filtration experiments revealed a peak of methylcitrate synthase distinct from the citrate synthase of the bacterium. The enzyme was induced by growth on propionate and on propanol and heptanoate, precursors of propionate. These results suggest thatP. aeruginosa may utilize the methylcitrate cycle to metabolize propionate, a pathway heretofore only described in the yeastCandida lipolytica.     

Induction ofent-kaurene biosynthesis by low temperature in dwarf peas

Germinating pea (Pisum sativum L.) seeds of two dwarf cultivars, “Progress No. 9” and “Green Arrow”, and two tall cultivars, “Alaska” and “Alderman”, were treated with low temperature (3–5°C) for 14 days and then transferred to normal growing conditions (19–21°C for 16 h/14.5–16.5°C for 8 h) for an additional 10 days. Biosynthesis of [¹⁴C]ent-kaurene from [¹⁴C]2-mevalonic acid (2-MVA) was assayed in cell-free enzyme extracts prepared from shoot tips 10 days after cold treatment and was compared with activity in enzyme extracts prepared from noncold-treated, 10-day-old control plants. Shoot lengths of cold-treated plants were measured throughout a 35-day period and compared with shoot lengths of plants grown without cold treatment for 25–35 days. Low temperature induced a five-to 10-fold enhancement ofent-kaurene, hence potentially gibberellin (GA), biosynthesis in seedlings of the two dwarf cultivars but not in the tall cultivars. However, the lack of an increase in growth rate in the cold-treated dwarfs indicated that endogenous GA biosynthesis remained blocked at some point beyondent-kaurene in the biosynthetic pathway. Since the late-flowering “Alderman” cultivar did not exhibit enhanced biosynthesis ofent-kaurene, it appears that if vernalization in late-flowering cultivars of peas is correlated with enhanced GA biosynthesis, it is not the early part of the biosynthetic pathway which is affected.     

Some pathogenic effects of a species ofPleistophora [Protozoa,Microsporida] forAgrotis exclamationis and other noctuids

Larvae of noctuids were inoculatedper os with spores of a species ofPleistophora isolated fromAgrotis exclamationis (L.). The mean median lethal dose for mainly 3rd instar larvae ofA. exclamationis was 1.38×10⁵ spores per larva accumulating 34 or 35 days after inoculation and the mean slope for the regression of mortality on dose was 0.82. Third instar larvae ofA. exclamationis andSpodoptera frugiperda (J.E. Smith) inoculated with 2.5×10⁴ spores gained weight quicker than uninfected ones until between 8 days (A. exclamationis) and 13 days (S. frugiperda) post-inoculation. Thereafter they grew slower than uninfected individuals. Correspondingly, the feeding rate of inoculated larvae ofA. exclamationis was greater than that of untreated ones until 14 days post-inoculation but thereafter was less. Larvae ofNoctua pronuba (L.)Phlogophora meticulosa L. andSpodoptera littoralis (Boisduval) but notAgrotis segetum (Denis & Schiffermüller) were also susceptible to infection. The species ofPleistophora considered here corresponds toP. schubergi noctuidae (Veremtchuk & Issi) in spore morphology, tissue specificity and host range, except that is was non-infective for the typehostA. segetum. It is probably insufficiently pathogenic for use in the biological control of noctuids.     

Catabolism of naphthalene and salicylate byPseudomonas fluorescens

Analysis of spent naphthalene growth media ofPseudomonas fluorescens by GC-MS revealed the presence of salicylate. Gentisate 1,2-dioxygenase and pyrocatechol 1,2-dioxygenase were induced by growth on naphthalene, whereas only pyrocatechol 1,2-dioxygenase was induced during growth on salicylate. These results suggest the existence of alternative degradative routes of salicylate,via gentisate and pyrocatechol, which are involved in the catabolism of naphthalene.     

Changes in nucleic acid synthesis in cotyledons and apical buds of chenopodium rubrum l. associated with photoperiodic induction

The nucleic acid (NA) fractions were analyzed in cotyledons and apical buds ofChenopodium rubrum plants by means of acrylamide electrophoresis at the end of the dark period of a different number of photoperiodic cycles or after transfer of the plants to light for 4 h subsequent to the termination of the dark period. The plants were labelled with³²P three hours prior to sampling. The uptake of³²P into the cotyledons was higher in light than in darkness in all cases, however, it was not in correlation with³²P incorporation into the NA fractions. After one dark period lasting 8 or 16 h NA synthesis in light did not increase in comparison with darkness. After two or more photoperiodic cycles NA synthesis was higher in light than in darkness irrespective of whether the dark period lasted 8 or 16 h. NA synthesis was distinctly highest after two inductive cycles lasting 16 h. In buds NA synthesis was slightly shifted in favour of ribosomal RNA as compared with cotyledons. In the cotyledons the increase in light was mainly duo to a raise of rRNA synthesis whereas in the buds synthesis of sRNA and DNA increased, as well.