Intrinsic uncoupling in the ATP synthase of Escherichia coli. Studies on WT and ε-truncated mutants

The H+/ATP ratio in the catalysis of ATP synthase has generally been considered a fixed parameter. However, Melandri and coworkers have recently shown that, in the ATP synthase of the photosynthetic bacterium Rb.capsulatus, this ratio can significantly decrease during ATP hydrolysis when the concentration of either ADP or Pi is maintained at a low level (Turina et al., 2004). The present work has dealt with the ATP synthase of E.coli, looking for evidence of this phenomenon of intrinsic uncoupling in this organism as well. First of all, we have shown that the DCCD-sensitive ATP hydrolysis activity of E.coli internal membranes was strongly inhibited by ADP and Pi, with a half-maximal effect in the submicromolar range for ADP and at 140 µM for Pi. In contrast to this monotonic inhibition, however, the proton pumping activity of the enzyme, as estimated under the same conditions by the fluorescence quenching of the ΔpH-sensitive probe ACMA, showed a clearly biphasic progression, both for Pi, increasing from 0 up to approximately 200 µM, and for ADP, increasing from 0 up to a few µM. We have interpreted these results as indicating that the occupancy of ADP and Pi binding sites shifts the enzyme from a partially uncoupled state to a fully coupled state, and we expect that the ADP- and Pi-modulated intrinsic uncoupling is likely to be a general feature of prokaryotic ATP synthases. Moreover, the biphasicity of the proton pumping data suggested that two Pi binding sites are involved. In order to verify whether the same behaviour could be observed in the isolated enzyme, we have purified the ATP synthase of E.coli and reconstituted it into liposomes. Similarly as observed in the internal membrane preparation, in the isolated and reconstituted enzyme it was possible to observe inhibition of the hydrolytic activity by ADP and Pi (with half-maximal effects at few µM for ADP and at 400 µM for Pi) with a concomitant stimulation of proton pumping. Both the inhibition of ATP hydrolysis and the stimulation of proton pumping as a function of Pi were lost upon ADP removal by an ADP trap. These data have made it possible to conclude that the results obtained in E.coli internal membranes are not due to the artefactual interference of enzymatic activities other than the ones of the ATP synthase. In addition, data obtained with liposomes have allowed a calibration of the ACMA signal by ΔpH transitions of known extent, leading to a quantitative evaluation of the proton pumping data. Finally, we have focused our efforts on searching for a possible structural candidate involved in the phenomenon of intrinsic uncoupling. The ε-subunit of the ATP-synthase is known as an endogenous inhibitor of the hydrolysis activity of the complex and appears to undergo drastic conformational changes between a non-inhibitory form (down-state) and an inhibitory form (up-state)(Rodgers & Wilce, 2000; Gibbons et al., 2000). In addition, the results of Cipriano & Dunn (2006) indicated that the C-terminal domain of this subunit played an important role in the coupling mechanism of the pump, and those of Capaldi et al. (2001), Suzuki et al. (2003) were consistent with the down-state showing a higher hydrolysis-to-synthesis ratio than the up-state. Therefore, we decided to search for modulation of pumping efficiency in a C-terminally truncated ε mutant. A low copy number expression vector has been built, carrying an extra copy of uncC, with the aim of generating an ε-overexpressing E.coli strain in which normal levels of assembly of the mutated ATP-synthase complex would be promoted. We have then compared the ATP hydrolysis and the proton pumping activity in membranes prepared from these ε-overexpressing E.coli strains, which carried either the WT ε subunit or the ε88-stop truncated form. Both strains yielded well energized membranes. Noticeably, they showed a marked difference in the inhibition of hydrolysis by Pi, this effect being largely lost in the truncated mutant. However, pre-incubation of the mutated enzyme with ADP at low nanomolar concentrations (apparent Kd = 0.7nM) restored the hydrolysis inhibition, together with the modulation of intrinsic uncoupling by Pi, indicating that, contrary to wild-type, during membrane preparation the truncated mutant had lost the ADP bound at this high-affinity site, evidently due to a lower affinity (and/or higher release) for ADP of the mutant relative to wild type. Therefore, one of the effects of the C-terminal domain of ε appears to be to modulate the affinity of at least one of the binding sites for ADP. The lack of this domain does not appear so much to influence the modulability of coupling efficiency, but instead the extent of this modulation. At higher preincubated ADP concentrations (apparent Kd = 117nM), the only observed effects were inhibition of both hydrolysis and synthesis, providing a direct proof that two ADP-binding sites on the enzyme are involved in the inhibition of hydrolysis, of which only the one at higher affinity also modulates the coupling efficiency.

Low codimensional intrinsic regular submanifolds in the Heisenberg group H^n

The thesis mainly concerns the study of intrinsically regular submanifolds of low codimension in the Heisenberg group H^n, called H-regular surfaces of low codimension, from the point of view of geometric measure theory. We consider an H-regular surface of H^n of codimension k, with k between 1 and n, parametrized by a uniformly intrinsically differentiable map acting between two homogeneous complementary subgroups of H^n, with target subgroup horizontal of dimension k. In particular the considered submanifold is the intrinsic graph of the parametrization. We extend various results of Ambrosio, Serra Cassano and Vittone, available for the case when k = 1. We prove that the uniform intrinsic differentiability of the parametrizing map is equivalent to the existence and continuity of its intrinsic differential, to the local existence of a suitable approximating family of Euclidean regular maps, and, when the domain and the codomain of the map are orthogonal, to the existence and continuity of suitably defined intrinsic partial derivatives of the function. Successively, we present a series of area formulas, proved in collaboration with V. Magnani. They allow to compute the (2n+2−k)-dimensional spherical Hausdorff measure and the (2n+2−k)-dimensional centered Hausdorff measure of the parametrized H-regular surface, with respect to any homogeneous distance fixed on H^n. Furthermore, we focus on (G,M)-regular sets of G, where G and M are two arbitrary Carnot groups. Suitable implicit function theorems ensure the local existence of an intrinsic parametrization of such a set, at any of its points. We prove that it is uniformly intrinsically differentiable. Finally, we prove a coarea-type inequality for a continuously Pansu differentiable function acting between two Carnot groups endowed with homogeneous distances. We assume that the level sets of the function are uniformly lower Ahlfors regular and that the Pansu differential is everywhere surjective.

Adapting fruit production to climate change: how to increase intrinsic fruit quality and save water through deficit irrigation scheduling.

Fruit crops are an important resource for food security, since more than being nutrient they are also a source of natural antioxidant compounds, such as polyphenols and vitamins. However, fruit crops are also among the cultivations threatened by the harmful effects of climate change This study had the objective of investigating the physiological effects of deficit irrigation on apple (2020-2021), sour cherry (2020-2021-2022) and apricot (2021-2022) trees, with a special focus on fruit nutraceutical quality. On each trial, the main physiological parameters were monitored along the growing season: i) stem and leaf water potentials; ii) leaf gas exchanges; iii) fruit and shoot growth. At harvest, fruit quality was evaluated especially in terms of fruit size, flesh firmness and soluble solids content. Moreover, it was performed: i) total phenolic content determination; ii) anthocyanidin concentration evaluation; and iii) untargeted metabolomic study. Irrigation scheduling in apricot, apple and sour cherry is surely overestimated by the decision support system available in Emilia-Romagna region. The water stress imposed on different fruit crops, each during two years of study, showed as a general conclusion that the decrease in the irrigation water did not show a straightforward decrease in plant physiological performance. This can be due to the miscalculation of the real water needs of the considered fruit crops. For this reason, there is the need to improve this important tool for an appropriate water irrigation management. Furthermore, there is also the need to study the behaviour of fruit crops under more severe deficit irrigations. In fact, it is likely that the application of lower water amounts will enhance the synthesis of specialized metabolites, with positive repercussion on human health. These hypotheses must be verified.