Moshe Reuveni

Antifungal activity of tobacco osmotin has specificity and involves plasma membrane permeabilization

Osmotin protein is able to inhibit in vitro the growth of a number of unrelated pathogens. A survey of 31 isolates representing 18 fungal genera indicated that sensitivity may be determined at the genus level. Hyphal growth of Aspergillus flavus, Aspergillus parasitica, Rhizoctonia solani and Macrophomina phaseolina was highly resistant to osmotin whereas the growth of Bipolaris, Fusarium and Phytophthora species was very sensitive. Of all fungi tested Trichoderma longibrachiatum hyphal growth was most inhibited by osmotin treatment. Osmotin either induced spore lysis, inhibited spore germination or reduced germling viability in seven fungal species that exhibited some degree of sensitivity in hyphal growth inhibition tests. The species-specific growth inhibition was correlated with the ability of osmotin to dissipate the fungal membrane pH gradient. Both growth inhibition and pH gradient dissipation by osmotin were sensitive to NaCl and other inorganic cations. Cells of T. longibrachiatum were insensitive to osmotin after plasmolysis, suggesting that the cell wall may be a component of the mechanism by which osmotin permeabilizes the plasma membrane and kills fungal cells.

Transcriptional Profiling of high pigment-2dg Tomato Mutant Links Early Fruit Plastid Biogenesis with Its Overproduction of Phytonutrients

Phenotypes of the tomato (Solanum lycopersicum) high pigment-2dg (hp-2dg) and hp-2j mutants are caused by lesions in the gene encoding DEETIOLATED1, a negative regulator of light signaling. Homozygous hp-2dg and hp-2j plants display a plethora of distinctive developmental and metabolic phenotypes in comparison to their normal isogenic counterparts. These mutants are, however, best known for the increased levels of carotenoids, primarily lycopene, and other plastid-accumulating functional metabolites. In this study we analyzed the transcriptional alterations in mature-green, breaker, and early red fruits of hp-2dg/hp-2dg plants in relation to their normal counterparts using microarray technology. Results show that a large portion of the genes that are affected by hp-2dg mutation display a tendency for up- rather than down-regulation. Ontology assignment of these differentially regulated transcripts revealed a consistent up-regulation of transcripts related to chloroplast biogenesis and photosynthesis in hp-2dg mutants throughout fruit ripening. A tendency of up-regulation was also observed in structural genes involved in phytonutrient biosynthesis. However, this up-regulation was not as consistent, positioning plastid biogenesis as an important determinant of phytonutrient overproduction in hp-2dg and possibly other hp mutant fruits. Microscopic observations revealed a highly significant increase in chloroplast size and number in pericarp cells of mature-green hp-2dg/hp-2dg and hp-2j/hp-2j fruits in comparison to their normal counterparts. This increase could be observed from early stages of fruit development. Therefore, the molecular trigger that drives phytonutrient overproduction in hp-2dg and hp-2j mutant fruits should be initially traced at these early stages.

Molecular Aspects of Anthocyanin fruit Tomato in Relation to high pigment-1

The tomato Anthocyanin fruit (Aft) genotype is characterized by purple color in skin and outer pericarp of its fruits due to higher levels of anthocyanins-flavonoid metabolites. Our objectives were to carry out metabolic and molecular characterization of this genotype, emphasizing its interaction with the high pigment-1 (hp-1) mutation, known to increase flavonoids in tomato fruits. These objectives fit the growing interest in developing tomato fruits with higher levels of functional metabolites. Our results show that 1) Aft fruits are also characterized by significantly higher levels of the flavonols quercetin and kaempferol, thus enhancing their functional value; 2) the tomato Anthocyanin1 (Ant1) gene, encoding a Myb transcription factor, displayed nucleotide and amino acid polymorphisms between the Aft genotype and cultivated genotypes; 3) a DNA marker based on Ant1 showed that the Aft trait is encoded by a single locus on chromosome 10 fully associated with Ant1; and 4) double homozygotes Aft/Aft hp-1/hp-1 plants displayed a more-than-additive effect on the production of fruit anthocyanidins and flavonols. This effect was manifested by approximately 5-, 19-, and 33-fold increase of petunidin, malvidin, and delphinidin, respectively, in the double mutants compared with the cumulative levels of their parental lines.

Differential inhibition by Bacillus thuringiensis δ endotoxin of leucine and aspartic acid uptake into BBMV from midgut of Manduca sexta

Pre-incubation of brush border membrane vesicles (BBMV) isolated from the midgut of Manduca sexta with activated Bacillus thuringiensis delta endotoxin for a short period resulted in differential inhibition of K(+)-dependent transport of leucine relative to the effect on K(+)-dependent transport of aspartic acid. The difference in I1/2 (5 fold greater for aspartic acid than for leucine) is interpreted as the result of enhanced binding of the B. thuringiensis delta endotoxin to the leucine transport system.

Leucine transport into brush border membrane vesicles from guts of Leptinotarsa decemlineata and Manduca sexta

Abstract 1. 1. Compared to crude homogenates, Manduca sexta brush border membrane vesicles (BBMV) had increased specific activity of marker enzymes leucine aminopeptidase and alkaline phosphatase while Leptinotarsa decemlineata BBMV had increased leucine aminopeptidase specific activity and decreased alkaline phosphatase specific activity. 2. 2. L. decemlineata BBMV were impermeable to a proton gradient while M. sexta BBMV sustained an alkali cation gradient. 3. 3. Both K + -stimulated and K + -independent unidirectional leucine transport pathways were identified in M. sexta BBMV while only cation-independent unidirectional leucine transport was identified in L. decemlineata BBMV.

β-Glucosidase activity is involved in scent production in Narcissus flowers

Abstract Extrinsic environmental cues and intrinsic developmental stages of the flower control the production of scent from flowers. Flowers emit scent only when they are open; yet, the precursors for the aromatic compounds are also present in buds, stored as non-fragrant glycosides in the vacuole. We demonstrate that in Narcissus flowers scent emission is concurrent with an increase in the activity of β-glucosidase. The inhibition in vivo of β-glucosidase activity decreases scent emission from Narcissus flowers. The β-glucosidase activity was partially purified and the Km, Vmax and inhibition by gluconic acid lactone was determined.

Characterization of the INTENSE PIGMENT tomato genotype emphasizing targeted fruit metabolites and chloroplast biogenesis.

The tomato INTENSE PIGMENT (IP) genotype is characterized by intense visual pigmentation of unripe and ripe fruits, not thoroughly analyzed thus far. This study was therefore designed to analyze key morphologic, metabolomic, and photomorphogenic phenotypes of this genotype in comparison to its near-isogenic normal counterpart and to evaluate its significance relative to other tomato mutants known for increased fruit pigmentation. The IP genotype produced smaller and darker red fruits, and a substantially increased chloroplast biogenesis was found in its green fruit and leaf tissues. Ripe-red fruits of the IP genotype produced 34-38% more soluble solids and up to 62.6% more carotenoids, but no differences were found in the concentration of flavonoid compounds in its peel tissue. The IP genotype was found to display a greater hypocotyl inhibition response to blue and yellow light, but a more prominent and novel response to total darkness. As a whole, the IP genotype exhibited highly desirable traits, making it a valuable genotype for tomato breeders attempting to introduce functional and taste qualities into tomato fruits.

Decrease in vacuolar pH during petunia flower opening is reflected in the activity of tonoplast H+-ATPase

Summary Flower color is determined by two major factors, pigments present in the vacuole, and intra-vacuolar environment (vacuolar pH and metal ion content). Several reports demonstrate the importance of vacuolar pH in determining flower color, and show that increase in vacuolar pH changes the color of the petal. We study vacuolar pH in petunia flower as a model system, and the H+ pumps (H+-ATPase and H+-PPase) using flower pH mutants. Two lines of petunia are being used in this study, a red line (R) with a low vacuolar pH (flower sap pH 5.6) and a purple line (P) with a high vacuolar pH (flower sap pH 6.1). ATP hydrolyzing activity of the tonoplast H+-ATPase of petals from the R line was three times higher than that from the P line. Western blot analysis showed similar amounts of the 70 kD and 58 kD subunits of vacuolar H+-ATPase in the two lines; however, subunit E amounts differed between the lines. The difference in the pH of the flower sap manifested with higher amounts of subunit E in the R line. The two lines did not differ in their organic acids synthesis. Genetic analysis of R and P lines showed that vacuolar pH is partially controlled by a single dominant gene (Ph gene).

Modification of Proton Transport Kinetics of the Plasma Membrane H+-ATPase after Adaptation of Tobacco Cells to NaCI

Summary Plasma membrane enriched vesicle fractions were isolated on discontinuous sucrose gradients from unadapted and 428 mM NaCl adapted tobacco cells ( Nicotiana tabacum L. var. Wisconsin 38). The fractions collected from the 36/45 % interfaces were determined to be plasma membrane enriched based on analyses of marker enzyme activities. Kinetic properties of H + -transport (determined by [ 14 C]-SCN - uptake) of the plasma membrane H + -ATPase of adapted cells were substantially different than those of unadapted cells. H + transport activity of the plasma membrane H + -ATPase from NaCl adapted cells was more susceptible to substrate inhibition by free ATP (k i = 3.1 mM) than the activity of the plasma membrane H + -ATPase from unadapted cells (k i = 10 mM). The plasma membrane H + -ATPase from NaCl adapted cells exhibited an apparent K m for MgATP of 0.6 ± 0.2 mM compared with 1.76 ± 0.5 mM for the plasma membrane H + -ATPase from unadapted cells. These data indicate that the plasma membrane H + -ATPase of adapted cells can function efficiently in saline environments where increased competition for the ATP pool is presumed.

Absorption pathways of amino acids in the midgut of Manduca sexta larvae

Abstract The unidirectional transport of leucine and aspartic acid by midgut epithelium cells of the tobacco ornworm, Manduca sexta , was investigated using brush border membrane vesicles. Both K + -stimulated and K + -insensitive transport pathways for these two amino acids were identified. Kinetic analyses and profiles of cis-inhibition by other amino acids demonstrated that leucine and aspartic acid are transported by separate pathways and exhibit different sensitivity to potassium ion.