Factors affecting variability in anther culture and in conversion of androgenic embryos ofSolanum phureja

The variation for embryo production in anther ofSolanum phureja was examined as a function of maximum greenhouse temperature prior to bud harvest and innate responsiveness among anthers within a bud. Four clones ofS. phuyreja were grown in a greenhouse under a 16-h photoperiod. The temperature was monitored continuously. Buds (60 per day on 10 days) were collected and the anthers cultured in two groups of five flasks (30 anthers per flask). In the first group, each flask contained the 30 anthers from six buds; in the second group, each flask contained one anther from each of 30 buds. Significantly smaller coefficients of variation were observed for the second group, suggesting that variation for embryogenic capcity among buds was greater than that among anthers within a bud. Variation in embryo yield as a function of greenhouse temperature was examined by stepwise regression analysis. Embryogenic capacity of one clone was adversely affected by high temperatures (31–37°C) that occurred two and seven days before bud harvest. However, similarly high temperatures appeared to enhance the androgenic response of another clone. Conversion of anther-derived embryos over three subcultures to fresh regeneration medium was examined as a function of anther donor or clone, cold pretreatment of embryos, and morphological classification of embryos. Only clonal origin significantly affected conversion rate which ranged from 12.5% to 46.0%. Conversion rate declined on each serial subculture.Abbreviations BA N⁶-benzyladenine - GA₃ gibberellic acid, IAA-indole-3-acetic acid     

Prosystemin,a prohormone that modulates plant defense barriers,is an intrinsically disordered protein

Prosystemin, originally isolated from Lycopersicon esculentum, is a tomato pro‐hormone of 200 aminoacid residues which releases a bioactive peptide of 18 aminoacids called Systemin. This signaling peptide is involved in the activation of defense genes in solanaceous plants in response to herbivore feeding damage. Using biochemical, biophysical and bioinformatics approaches we characterized Prosystemin, showing that it is an intrinsically disordered protein possessing a few secondary structure elements within the sequence. Plant treatment with recombinant Prosystemin promotes early and late plant defense genes, which limit the development and survival of Spodoptera littoralis larvae fed with treated plants.     

Formation of (-⊃-1′,2′-epi-2-cis-xanthoxin acid from a precursor of abscisic acid

Tomato shoots and avocado mesocarp supplied with (±)-[2-¹⁴C]-5-(1,2-epoxy-2,6,6-trimethylcyclohexyl)-3-methylpenta-cis-2-trans-4-dienoic acid metabolize it into (+)-abscisic acid and a more polar material that was isolated and identified as (?)-epi-1′(R),2′(R)-4′(S)-2-cis-xanthoxin acid. The (+)-1′(S),2′(S)-4′(S)-2-cis-xanthoxin acid recently synthesized from natural violaxanthin, has the 1′,2′-epoxy group on the opposite side of the ring to that of the 4′(S)-hydroxyl group and the compound is rapidly converted into (+)-abscisic acid. The 1′,2′-epoxy group of (?)-1′,2′-epi-2-cis-xanthoxin acid is on the same side of the ring as the 4′(S) hydroxyl group: the compound is not metabolized into abscisic acid. The configuration of the 1′,2′-epoxy group probably controls whether or not the 4′(S) hydroxyl group can be oxidized. (+)-2-cis-Xanthoxin acid is probably not a naturally occurring intermediate because a ‘cold trap’, added to avocado fruit forming [¹⁴C]-labelled abscisic acid from [2-¹⁴C]mevalonate, failed to retain [¹⁴C] label.     

Crystal structure of nonspecific lipid transfer protein from Solanum melongena

Lipids are considered to protect protein allergens from proteolysis and are generally seen to exist in a bound form. One of the well‐known plant protein families with bound lipids is non‐specific lipid transfer proteins (nsLTPs). Structure‐function relationships in the case of the members of non‐specific lipid transfer protein family are not clearly understood. As part of exploring the seed proteome, we have analyzed the proteome of a member of Solanaceae family, Solanum melongena (eggplant) and a non‐specific lipid transfer protein from S. melongena, SM80.2 was purified, crystallized and the structure was determined at 1.87 Å resolution. Overall, the tertiary structure is a cluster of α‐helices forming an internal hydrophobic cavity. Absence of conserved Tyr79, known to govern the plasticity of hydrophobic cavity, and formation of hydrogen bond between Asn79 and Asn36 further reduced the pocket size. Structural analysis of SM80.2 thus gives insight about a new hydrogen bond mediated mechanism followed in closure of the binding pocket. Extra electron densities observed at two different places on the protein surface and not in the cavity could provide interesting physiological relevance. In light of allergenic properties, probably overlapping of epitopic region and ligand binding on surface could be a main reason. This work shows first crystal structure of A‐like nsLTP with a close binding pocket and extra density on the surface suggesting a plausible intermediate state during transfer.     

The stereochemistry of cyclization in abscisic acid

The hydroxylation of the pro-6′-(R)-methyl of (+)-abscisic acid, which then cyclises to phaseic acid, was used to define the origin in mevalonate of the 6′-methyl groups. Abscisic acid (ABA), biosynthesised from [2-¹⁴C, 2-³H₂]-mevalonate, was metabolized to phaseic acid by tomato shoots. The slight loss of [³H] from the phaseate, and to a lesser extent from the ABA, suggested that the unlabelled 6′-methyl was hydroxylated. This was confirmed by Kuhn-Roth oxidation of methyl phaseate to give [¹⁴C, ³H]-acetate. The data also suggest that ABA is converted to dihydrophaseate via free phaseate, the conjugates being formed from each free acid.     

Effect of ageing on sterol metabolism in potato tuber slices

When fresh potato tuber slices were incubated with [1-¹⁴C]-sodium acetate, cycloartenol was heavily labelled but no radioactivity was recovered in 24-methylene cycloartanol and free sterols. If potato slices were aged for 0–24 hr before feeding with radioactive acetate, a rapid increase of the label in the sterol precursors and the free sterols was observed. The free sterol content was 5 × higher after ageing for 24 hr. Isofucosterol synthesis was especially stimulated. The synthesis of sterols during the ageing process seems to be related to the appearance of a cycloartenol C24-methylase and may be linked to a biogenesis of membranes.Nomenclature: (1) 4,4,14α-trimethyl 9β, 19β-cyclo-5α-cholest-24-en 3β-ol; (2) 4,4,14α-trimethyl 9β, 19β-cyclo-5α-ergost-24(28)-en 3β-ol; (3) 4α,14α-dimethyl 9β,19β-cyclo 5α-ergost 24(28)-en 3β-ol; (4) 4α, 14α-dimethyl 5α-ergosta 8.24(28)-dien 3β-ol; (5) 4α-methyl 5α-ergosta 7,24(28)-dien 3β-ol; (6) ergosta 5,24(28)-dien 3β-ol; (7) stigmasta 5,Z-24(28)-dien 3β-ol; (8) (24R)-24 methyl cholest 5-en 3β-ol; (9) (24R)-24 ethyl cholest 5-en 3β-ol; (10) (24S)-24 ethyl cholesta 5,E-22(23)-dien 3β-ol; (11) cholest 5-en 3β-ol.     

Effectiveness of GF‐120NF Fruit Fly Bait as a suppression tool for Bactrocera latifrons (Diptera: Tephritidae)

Bactrocera latifrons (Hendel) is a tephritid fruit fly of primarily Asian distribution that has invaded Hawaii and, more recently, the continent of Africa (Tanzania and Kenya). It primarily infests solanaceous fruits, so has the potential to impact production of crops such as peppers (Capsicum annuum L. and Capsicum frutescens L.), eggplant (Solanum melongena L.), African eggplant (Solanum aethiopicum L.) and tomatoes (Solanum lycopersicum L.). Because little work has been done to develop suppression techniques for this fruit fly species, field cage tests of the effectiveness of a commercially available bait spray, GF‐120NF Fruit Fly Bait, against wild B. latifrons were conducted. Sexually mature B. latifrons adults (75 male and 75 female) were introduced to both a control cage and a treatment cage, each of which held six fruiting Anaheim chili pepper (C. annuum L.) plants. Fruits were harvested, and assessed for infestation, both before and after the application of the bait spray in the treatment cage. There was no difference in infestation rate between control and treatment cages before the application of the bait spray, whereas there was a significantly lower infestation rate in treatment cages following the application of the bait spray. Post‐spray infestation rate in the treatment cages (in two separate, replicated bioassays) was always zero and no live flies were detected in the treatment cages at the end of the trials. The results of this study provide evidence that GF‐120NF Fruit Fly Bait should be effective in suppressing B. latifrons populations in the field.     

Selection of atrazine-resistant plants by <Emphasis Type="Italic">in vitro</Emphasis> mutagenesis in pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis>)

The effects of atrazine on cotyledon cultures of Capsicum annuum (L.) cv. G₄ were investigated with a view of establishing a system for in vitro selection of resistant mutants. At low levels of herbicide produced little growth inhibition, some chlorophyll loss occurred associated with the production of albino shoots. At 20 mg l⁻¹ atrazine bleaching was more pronounced and was accompanied by the development of necrotic spots; however, efficient bleaching was associated with severe suppression of growth. Mutagenized cotyledon explants resulted in production of herbicide-resistant plants on medium containing selective levels of sucrose (0.5%) and atrazine (20 mg l⁻¹). Differential morphogenetic responses were observed when the levels of sucrose (0.5–5%) were altered. Shoot regeneration was maximum in 2 sucrose and the regenerating ability decreased with a further increase in sucrose concentration (3%–5%). However, lowering of sucrose concentration from 2 to 0.5% caused complete bleaching of explants and permitted the selection of herbicide-resistant plants. Complete atrazine-resistant plantlets were obtained after rooting of regenerated green shoots on rooting medium containing 10 mg l⁻¹atrazine, 1.0 mg l⁻¹IAA and 0.5% sucrose. Leaf-segment assay of differentiated plants revealed that all regenerants were resistant to the atrazine. Reciprocal crosses between atrazine-resistant and -sensitive plants showed a non-Mendelian transmission of resistance trait.     

γ-Glutamylcyclotransferase in tobacco suspension cultures: Catalytic properties and subcellular localization

Steinkamp, R., Schweihofen, B. and Rennenberg, H. 1987. γ-Glutamylcyclotransfer-ase in tobacco suspension cultures: Catalytic properties and subcellular localization.
γ-Glutamylcyclotransferase activity (EC 2.3.2.4) in ammonium sulfate precipitates (40–70% saturation) of extracts from cultured tobacco cells ( Nicoliana tabacum L. cv. Samsun) was analyzed as liberation of 5-oxo-proline from L-γ-glutamyl dipeptides. The enzyme was highly specific for the sulfur containing γ-glutamyl dipeptides γ-glutamyl-L-methionine and γ-glutamyl-i.-cysteine. Maximum activity was obtained at pH 8.7 and 35°C. As also observed with animal γ-glutamylcyclotransferase, the tobacco enzyme exhibited a relatively low substrate affinity (γ-glutamyl-i.-methionine: K_m 2.2 ± 0.4 mM ). In contrast to animal γ-glutamylcyclotransferase, the tobacco enzyme was not inhibited by the D-isomerof the substrate D-γ-glutamyl-i.-methionine; it also did not use the D isomer as a substrate. γ-Glutamylcyclotransferase of tobacco cells was shown to be a soluble enzyme entirely localized in the cytoplasm.     

γ-Glutamyl-transpeptidase in tobacc suspension cultures: Catalytic properties and subcellular localization

γ-Glutamyl-transpeptidase activity (EC 2.3.2.2) was found in ammonium sulfate precipitates of extracts from cultured cells of Nicotiana tabacum L. var. Samsun. Specific activity up to 3.2 nmol (mg protein)⁻¹ min⁻¹ was achieved, using the artificial substrate γ-glutamyl- p -nitroanilide (K_m 0.6 m M ) instead of glutathione. Optimal enzyme activity was obtained at pH 8.0–8.5 and 45°C. The enzyme reaction was inhibited competitively by γ-glutamyl analogs (6-diazo-5-oxo-L-norleucine: K_i 0.76 μ M ; L-azaserine: K_i 0.23 m M ) or the inorganic ion m -periodate (K_i 0.43 m M ). Cell fractionation and in vivo experiments revealed that 77% of the γ-glutamyl-transpeptidase activity is localized in the soluble cytoplasmic fraction, while 20–23% of the enzyme is found on the outer surface of the plasmalemma.