With chlorophyll pigments from prolamellar bodies to light-harvesting complexes

The biosynthetic chain leading from 5-aminolevulinic acid to chlorophyll is localised to the plastid. Many of the enzymes are nuclear-encoded. NADPH-protochlorophyllide oxidoreductase (EC 1.3.1.33) is one such enzyme which is encoded by two different genes and can exist in an A and a B form. Its import into the plastid seems to be facilitated when protochlorophyllide is present in the chloroplast envelope. Within the plastid the reductase is assembled to thylakoids or prolamellar bodies. The specific properties of the reductase together with the specific properties of the lipids present in the etioplast inner membranes promote the formation of the three-dimensional regular network of the prolamellar bodies. The reductase forms a ternary complex with protochlorophyllide and NADPH that gives rise to different spectral forms of protochlorophyllide. Light transforms protochlorophyllide into chlorophyllide and this photoreaction induces a conformational change in the reductase protein which leads to a process of disaggregation of enzyme, pigment aggregates and membranes, which can be followed spectroscopically and with electron microscopy. The newly formed chlorophyllide is esterified by a membrane-bound nuclear-encoded chlorophyll synthase and the chlorophyll molecule is then associated with proteins into active pigment protein complexes in the photosynthetic machinery.     

Control of plant disease by perturbation of fungal polyamine metabolism

The diamine putrescine and the polyamines spermidine and spermine are ubiquitous in nature and are essential for cell proliferation. Since polyamine biosynthesis in plants can start from either ornithine or arginine, while fungal polyamine biosynthesis appears to utilise only the ornithine route, it was suggested that specific inhibition of fungal polyamine biosynthesis should be lethal. Indeed, inhibitors of polyamine biosynthesis, e.g. the ornithine decarboxylase inhibitor α-difluoromethylornithine, have been shown to inhibit fungal growth in vitro and to control fungal infections on a variety of plants under glasshouse and field conditions. It is now known that polyamine analogues can perturb polyamine metabolism leading to powerful antiproliferative effects in cancer cells. This paper reviews the results of a research programme focused on the synthesis and evaluation of putrescine analogues as novel fungicides. A number of aliphatic, alicyclic and cyclic diamines have been shown to possess considerable fungicidal activity, but although many of these compounds perturb polyamine metabolism in fungal cells, such changes are not considered sufficient to account for the observed antifungal effects. More recent work on spermidine analogues is also described.     

Elicitor-mediated induction of anthraquinone biosynthesis and regulation of isopentenyl diphosphate isomerase and farnesyl diphosphate synthase activities in cell suspension cultures of Cinchona robusta How.

Treatment of a Cinchona robusta How. cell suspension culture with a homogenate of Phytophthora cinnamomi resulted in cessation of growth and a rapid induction of the biosynthesis of anthraquinone-type phytoalexins. The strongest induction of anthraquinone biosynthesis was obtained when the elicitor was added in the early growth phase of the growth cycle. The accumulation of anthraquinones was accompanied by a tri-phasic response in the activity of isopentenyl diphosphate (IPP) isomerase (EC 5.3.3.2): phase I was characterised by a rapid induction of activity, reaching a maximum at 12 h after elicitation. During phase II, IPP isomerase rapidly decreased to levels below those found in untreated cells. At phase III, IPP isomerase activity increased again, reaching a second maximum at about 72 h after elicitation. During phase I, the activity of farnesyl diphosphate synthase (EC 2.5.1.10) was found to be suppressed. Extraction and assay conditions were optimised for IPP isomerase. The presence of Mn²⁺ in the incubation buffer resulted in a marked increase in the activity of the enzymes obtained from cells in phase I. The induction of IPP isomerase in combination with a concomitant inhibition of farnesyl diphosphate synthase might result in an efficient channeling of C₅-precursors into phytoalexin biosynthesis. Received: 23 August 1996 / Accepted: 20 March 1997     

Discrimination between cultivars of Vitis vinifera based on molecular variability concerning 5″ untranslated regions of the StSy-CHS genes

 The degree of polymorphism present in 5′ untranslated regions of stilbene synthase (StSy)-like loci was assessed. A ligation-mediated polymerase chain reaction (LM-PCR) cloning strategy was adopted to isolate sequences located immediately upstream of StSy coding regions. Among several clones, 13 randomly chosen fragments were analyzed at the sequence level. Four of the analyzed fragments appeared of particular interest. Two carried sequences reminiscent of micro-satellites, while the remaining fragments contained direct repeats. Oligonucleotides constructed against the specific DNA sequence of these clones disclosed a complex banding pattern when used in polymerase chain reaction (PCR)-analysis of 22 ancient varieties of grapevine. A total of 40 polymorphic bands could be identified and used to calculate coefficients of genetic similarity (GS) between varieties. GS values were used in cluster analysis to differentiate the 22 varieties. The data obtained are in good agreement with available information concerning the relationships between the varieties considered. This suggests the use of the method we have developed in fingerprinting studies of Vitis vinifera germ plasma. Received: 11 April 1996 / Accepted: 14 March 1997     

The cryptochrome family of photoreceptors

CRY1, the primary photoreceptor responsible for blue light-mediated inhibition of hypocotyl elongation in Arabidopsis thaliana, has been characterized. The properties of CRY1, and those of the related protein CRY2, and their relationship to the photolyase family of flavoproteins are discussed.     

Analysis of the chloroplast protein complexes by blue-native polyacrylamide gel electrophoresis (BN-PAGE)

Blue-native polyacrylamide gel electrophoresis (BN-PAGE) is a powerful procedure for the separation and characterization of the protein complexes from mitochondria. Membrane proteins are solubilized in the presence of aminocaproic acid and n-dodecylmaltoside and Coomassie-dyes are utilized before electrophoresis to introduce a charge shift on proteins. Here, we report a modification of the procedure for the analysis of chloroplast protein complexes. The two photosystems, the light-harvesting complexes, the ATP synthase, the cytochrome b ₆ f complex and the ribulose-bisphosphate carboxylase/oxygenase are well resolved. Analysis of the protein complexes on a second gel dimension under denaturing conditions allows separation of more than 50 different proteins which are part of chloroplast multi-subunit enzymes. The resolution capacity of the blue-native gels is very high if compared to 'native green gel systems' published previously. N-terminal amino acid sequences of single subunits can be directly determined by cyclic Edman degradation as demonstrated for eight proteins. Analysis of chloroplast protein complexes by blue-native gel electrophoresis will allow the generation of 'protein maps' from different species, tissues and developmental stages or from mutant organelles. Further applications of blue-native gel electrophoresis are discussed.     

Control of Trypanosoma cruzi Epimastigote Motility through the Nitric Oxide Pathway

ABSTRACT. Trypanosoma cruzi epimastigote motility can be enhanced by addition of L-arginine, to the culture. This effect is blocked by N-methyl-L-arginine, a competitive inhibitor of the nitric oxide synthase. N-methyl-D-aspartate and L-glutamate, two agonists of the NMDALglutamate receptor, also enhanced motility. This stimulation is blocked by MK-801 a noncompetitive antagonist of the NMDA receptor. In addition, sodium nitroprusside, a guanylyl cyclase stimulator and 8-Br-cyclic GMP, an analog of cyclic GMP, also stimulated epimastigote motility. It is suggested that an increase of intracellular cyclic GMP levels mediated by nitric oxide may be responsible for the increase in epimastigote motility.