A comparative study of melting and non-melting flesh peach cultivars reveals that during fruit ripening endo-polygalacturonase (endo-PG) is mainly involved in pericarp textural changes, not in firmness reduction.

Peach softening is usually attributed to the dismantling of the cell wall in which endo-polygalacturonase (endo-PG)-catalysed depolymerization of pectins plays a central role. In this study, the hypothesis that the function of endo-PG is critical for achieving a melting flesh fruit texture but not for reducing fruit firmness was tested by comparing pericarp morphology and endo-PG expression and localization in melting (MF) and non-melting flesh (NMF) fruit at successive stages of ripening. MF Bolero, Springbelle, and Springcrest, and NMF Oro-A and Jonia cultivars were analysed. Both MF and NMF fruit were left to ripen on the tree and reached a firmness of <10 Newtons (N). The image analysis of pericarp tissues revealed that during softening the loss of cell turgidity was a process common to mesocarp cells of all MF and NMF fruit and was clearly visible in peaches with a firmness of less than ∼20 N. In contrast, the loss of cell adhesion was a feature exclusively observed in ripe MF fruit pericarp. In this ripe fruit, large numbers of endo-PG isoforms were highly expressed and the enzyme localization corresponded to the middle lamella. As a consequence, wide apoplastic spaces characterized the pericarp of ripe MF peaches. In contrast, no loss of cell adhesion was observed in any NMF fruit or in unripe MF peaches. Accordingly, no endo-PG was detected in unripe NMF fruit, whereas few and poorly expressed enzyme isoforms were revealed in ripe NMF and in unripe MF peaches. In this fruit, the poorly expressed endo-PG localized mainly in vesicles within the cytoplasm and inner primary cell wall. On the whole the results suggested that endo-PG function was needed to achieve melting flesh texture, which was characterized by wide apoplastic spaces and partially deflated mesocarp cells. Conversely, endo-PG activity had no critical influence on the reduction of fruit firmness given the capacity of NMF peaches to soften, reaching values of 5-10 N. As in tomato, the change of symplast/apoplast water status seems to be the main process through which peach fruit regulates its firmness.

Changes in endopolygalacturonase levels and characterization of a putative endo-PG gene during fruit softening in peach genotypes with nonmelting and melting flesh fruit phenotypes.

The changes in endopolygalacturonase (endo-PG) levels and endo-PG expression in nonmelting flesh (NMF) and melting flesh (MF) peach fruits (Prunus persica) during softening were studied. The endo-PG gene was analysed to identify polymorphisms exploitable for early marker-assisted selection (MAS) of flesh texture. The role of endo-PG in softening was assessed by western and northern blotting and by biochemical analyses. Polymorphisms in the endo-PG gene were revealed by reverse transcription-polymerase chain reaction (RT-PCR) and sequencing. An endo-PG protein was detected in both NMF and MF fruits. The levels of this endo-PG protein were higher and increased with softening in MF fruits, but remained lower and were constant in NMF fruits. The different levels of endo-PG appeared to be caused by the differential expression of an endo-PG gene, whose open-reading frame (ORF) showed five single nucleotide polymorphisms (SNPs) in NMF 'Oro A' compared with MF 'Bolero'. One of these SNPs allowed us to determine the allelic configuration at the melting flesh (M) locus and also seemed to be exploitable for early MAS in other NMF/MF phenotypes. The NMF phenotype does not seem to be caused by a large deletion of the endo-PG gene.

Effects of deuterium oxide on growth, proton extrusion, potassium influx, and in vitro plasma membrane activities in maize root segments.

Elongation of subapical segments of maize (Zea mays) roots was greatly inhibited by (2)H(2)O in the incubation medium. Short-term exposure (30 min) to (2)H(2)O slightly reduced O(2) uptake and significantly increased ATP levels. (2)H(2)O inhibited H(+) extrusion in the presence of both low (0.05 mm) and high (5 mm) external concentrations of K(+) (about 30 and 53%, respectively at 50% [v/v] (2)H(2)O). Experiments on plasma membrane vesicles showed that H(+)-pumping and ATPase activities were greatly inhibited by (2)H(2)O (about 35% at 50% [v/v] (2)H(2)O); NADH-ferricyanide reductase and 1,3-beta-glucan synthase activities were inhibited to a lesser extent (less than 15%). ATPase activities present in both the tonoplast-enriched and submitochondrial particle preparations were not affected by (2)H(2)O. Therefore, the effect of short incubation time and low concentration of (2)H(2)O is not due to a general action on overall cell metabolism but involves a specific inhibition of the plasma membrane H(+) -ATPase. K(+) uptake was inhibited by (2)H(2)O only when K(+) was present at a low (0.05 mm) external concentration where absorption is against its electrochemical potential. The transmembrane electric potential difference (E(m)) was slightly hyperpolarized by (2)H(2)O at low K(+), but was not affected at the higher K(+) concentrations. These results suggest a relationship between H(+) extrusion and K(+) uptake at low K(+) external concentration.

Changes in the Levels of Calmodulin and of a Calmodulin Inhibitor in the Early Phases of Radish (Raphanus sativus L.) Seed Germination: Effects of Aba and Fusicoccin.

An inhibitor of Ca(2+)-calmodulin (Cam)-dependent brain phosphodiesterase was present in the soluble fraction of embryo axes from ungerminated radish (Raphanus sativus L.) seeds. This inhibitor is a Ca(2+)-dependent, Cam-binding protein; in fact: (a) its effect was strongly reduced by treatment with proteases; (b) the inhibition was counteracted by Cam but not by Ca(2+); (c) on gel filtration in the presence of Ca(2+), Cam co-chromatographed with the inhibitor. The inhibitor is heat stable and positively charged at pH 7.5. During early phases of germination, the fresh weight and the levels of DNA and RNA of embryo axes increased, the level of the inhibitor decreased, and the level of Cam increased. Abscisic acid (ABA) inhibited germination, the decrease of inhibitor, and the increase of Cam. Fusicoccin (FC) stimulated the increase in fresh weight but not the increase in the RNA and DNA levels; in this condition, the inhibitor level decreased and the increase in Cam level was higher than in the control. In the presence of both ABA and FC, there was an increase in fresh weight not accompanied by an increase in DNA and RNA levels; Cam increased and, on a fresh weight basis, reached the value of the control. These results indicate that the Ca(2+)-Cam system was activated in early germination of radish seeds by an increase in Cam and a decrease in the inhibitor levels, that FC, probably through the activation of membrane functions, increased Cam level, and that the ABA inhibition on germination was not mediated by the Ca(2+)-Cam system.

The separation of proteins based on their age, for the study of protein degradation in Escherichia coli.

Density labelling with 2H2O has been used in association with isopycnic centrifugation to isolate proteins of known age from cultures of Escherichia coli. The physical properties of protein samples of known age have been examined to detect a correlation between specific properties and susceptibility to degradation. No evidence of a correlation between size, charge, thermodynamic properties or amino acid composition, on one hand, and susceptibility to degradation, on the other hand, was observed following step-down conditions of growth. However, the SH content of proteins in E. coli does appear to be correlated with their susceptibility to degradation.

Effect of polar lipids on ATPase activity of membrane preparations from germinating radish seeds.

Membrane preparation (sedimenting between 13,000(g) and 80,000(g)) of germinating radish seeds (Raphanus sativus L.) was active in hydrolyzing ATP and, to a lesser extent, a variety of other phosphorylated compounds. Dicyclohexylcarbodiimide (DCCD) and diethylstilbestrol significantly inhibited the ATPase activity (40%) while their effect on hydrolysis of other phosphorylated compounds was much less.The sucrose density gradient analysis of the membrane preparation showed that the position of the DCCD-sensitive K(+)-dependent ATPase was similar to that found for plasma membrane of other plant material.Cholate treatment of membrane preparation removes almost all phospholipids, and ATPase activity is barely detectable. However, the addition of polar lipids completely restores the ATPase activity but does not restore general phosphatase activity.The ATPase of the polar lipids restored cholate preparation, showed a high sensitivity to DCCD and diethylstilbestrol (up to 90% inhibition), a complete dependence on Mg(2+), and a strong dependence on K(+) at low concentration; the pH optimum of ATPase was close to 6.5, and the K(m) for ATP-Mg was 0.51 millimolar. ATPase activity was much greater when polar lipids from 24-hour-germinated seeds were added.

Nuclear inheritance of resistance to antimycin A in Saccharomyces cerevisiae.

A group of 30 independent mutants of Saccharomyces cerevisiae, resistant to the respiratory inhibitor antimycin A, was investigated from a genetical and biochemical point of view. All the mutants can be grouped into two nuclear loci: AMY1 maps on the VII chromosome, between leu 1 and trp 5; AMY2 is close to its centromere on either chromosome XVIII or XIX. Both genes do not affect mitochondrial structures or functions.

Effects of abscisic acid, gibberellic acid and fusicoccin on the transmembrane potential during the early phases of germination in radish (Raphanus sativus L.) seeds.

During germination, the transmembrane electric potential (PD) of cortical cells of the embryonal axis of radish seeds (Raphanus sativus L.) rises from-120 mV initially to a maximum of-150 mV after 5 h incubation, then falls again to stable values of around-120 mV. Treatments inhibiting germination block the transitory PD increase. Administration of uncoupling agents or low temperatures, during the process of germination, produces a marked fall of the PD transitory increase. Abscisic Acid has a parallel inhibitory effect on PD and germination, while fusicoccin produces a rise in both; administration of abscisic acid with fusicoccin inhibits germination, while the PD remains at the high levels given by fusicoccin. These results are discussed in relation to ion exchange at membrane level.

Oxygen pressure, fatty acid composition and ergosterol level in Rhodotorula gracilis.

Cells of Rhodotorula gracilis grown for 6 hrs at 2 mm Hg O2 pressure when compared with cells grown for 6 hrs at 140 mm Hg, show: a) a large decrease in the level of ergosterol, b) a significant increase in the level of oleic acid and a decrease in the levels of linoleic and linolenic acids, both in fatty acid fraction of the phospholipids and in the free fatty acids and neutral fat fractions. The results suggest that the dehydrogenation of oleic acid to linoleic acid is preferentially inhibited at low O2 pressure. The possibility is considered that these changes of lipid metabolism might be causally related with decrease of the growth rate observed at low O2 pressure.

Adaptation of the mitochondrial systems of Rhodotorula gracilis to low oxygen pressure.

The obligate aerobic yeast, Rhodotorula gracilis, was grown in a liquid minimal medium at 1 mm Hg partial pressure of oxygen, conditions under which growth (measured as the increase in total protein) is reduced to 30% of the maximum rate. A significant increase in the ratio between mitochondrial oxidative enzymes and total protein occurs rapidly under these conditions. A concurrent increase in the ratio area of mitochondria/area of cytoplasm was also observed. The relative increase in mitochondrial enzymes, oxidase activities and mitochondrial membranes is due to the inhibition affecting the increase in cytoplasmic structures more significantly than the increase in mitochondrial structures at low pO2. The difference between mitochondrial and cytoplasmic syntheses cannot be ascribed to changes in the availability of ATP but it might rest with some other oxygen-utilising process (pyrimidine redox coenzymes, synthesis of sterols). The experimental conditions studied appear to offer a valuable tool for the investigation of the relationships between mitochondrial and cytoplasmic structures.