Valentino Casolo

The mitochondrial permeability transition pore (PTP) — An example of multiple molecular exaptation?

The mitochondrial permeability transition (PT) is a well-recognized phenomenon that allows mitochondria to undergo a sudden increase of permeability to solutes with molecular mass ≤ 1500Da, leading to organelle swelling and structural modifications. The relevance of PT relies on its master role in the manifestation of programmed cell death (PCD). This function is performed by a mega-channel (in some cases inhibited by cyclosporin A) named permeability transition pore (PTP), whose function could derive from the assembly of different mitochondrial proteins. In this paper we examine the distribution and characteristics of PTP in mitochondria of eukaryotic organisms so far investigated in order to draw a hypothesis on the mechanism of its evolution. As a result, we suggest that PTP may have arisen as a new function linked to a multiple molecular exaptation of different mitochondrial proteins, even though they could nevertheless still play their original role. Furthermore, we suggest that the early appearance of PTP could have had a crucial role in the establishment of endosymbiosis in eukaryotic cells, by the coordinated balancing of ATP production by glycolysis (performed by the primary phagocyte) and oxidative phosphorylation (accomplished by the endosymbiont). Indeed, we argue on the possibility that this new energetic equilibrium could have opened the way to the subsequent evolution toward metazoans.

The role of mild uncoupling and non-coupled respiration in the regulation of hydrogen peroxide generation by plant mitochondria

The roles of mild uncoupling caused by free fatty acids (mediated by plant uncoupling mitochondrial protein (PUMP) and ATP/ADP carrier (AAC)) and non‐coupled respiration (alternative oxidase (AO)) on H2O2 formation by plant mitochondria were examined. Both laurate and oleate prevent H2O2 formation dependent on the oxidation of succinate. Conversely, these free fatty acids (FFA) only slightly affect that dependent on malate plus glutamate oxidation. Carboxyatractylate (CAtr), an inhibitor of AAC, completely inhibits oleate‐ or laurate‐stimulated oxygen consumption linked to succinate oxidation, while GDP, an inhibitor of PUMP, caused only a 30% inhibition. In agreement, CAtr completely restores the oleate‐inhibited H2O2 formation, while GDP induces only a 30% restoration. Both oleate and laurate cause a mild uncoupling of the electrical potential (generated by succinate), which is then followed by a complete collapse with a sigmoidal kinetic. FFA also inhibit the succinate‐dependent reverse electron transfer. Diamide, an inhibitor of AO, favors the malate plus glutamate‐dependent H2O2 formation, while pyruvate (a stimulator of AO) inhibits it. These results show that the succinate‐dependent H2O2 formation occurs at the level of Complex I by a reverse electron transport. This generation appears to be prevented by mild uncoupling mediated by FFA. The anionic form of FFA appears to be shuttled by AAC rather than PUMP. The malate plus glutamate‐dependent H2O2 formation is, conversely, mainly prevented by non‐coupled respiration (AO).

Rooting depth, water relations and non-structural carbohydrate dynamics in three woody angiosperms differentially affected by an extreme summer drought

In 2012, an extreme summer drought induced species-specific die-back in woody species in Northeastern Italy. Quercus pubescens and Ostrya carpinifolia were heavily impacted, while Prunus mahaleb was largely unaffected. By comparing seasonal changes in isotopic composition of xylem sap, rainfall and deep soil samples, we show that P. mahaleb has a deeper root system than the other two species. This morphological trait allowed P  mahaleb to maintain higher water potential (Ψ), gas exchange rates and non-structural carbohydrates content (NSC) throughout the summer, when compared with the other species. More favourable water and carbon states allowed relatively stable maintenance of stem hydraulic conductivity (k) throughout the growing season. In contrast, in Quercus pubescens and Ostrya carpinifolia, decreasing Ψ and NSC were associated with significant hydraulic failure, with spring-to-summer k loss averaging 60%. Our data support the hypothesis that drought-induced tree decline is a complex phenomenon that cannot be modelled on the basis of single predictors of tree status like hydraulic efficiency, vulnerability and carbohydrate content. Our data highlight the role of rooting depth in seasonal progression of water status, gas exchange and NSC, with possible consequences for energy-demanding mechanisms involved in the maintenance of vascular integrity.

Mitochondrial bioenergetics linked to the manifestation of programmed cell death during somatic embryogenesis of Abies alba

The present work reports changes in bioenergetic parameters and mitochondrial activities during the manifestation of two events of programmed cell death (PCD), linked to Abies alba somatic embryogenesis. PCD, evidenced by in situ nuclear DNA fragmentation (TUNEL assay), DNA laddering and cytochrome c release, was decreased in maturing embryogenic tissue with respect to the proliferation stage. In addition, the major cellular energetic metabolites (ATP, NAD(P)H and glucose-6-phosphate) were highered during maturation. The main mitochondrial activities changed during two developmental stages. Mitochondria, isolated from maturing, with respect to proliferating cell masses, showed an increased activity of the alternative oxidase, external NADH dehydrogenase and fatty-acid mediated uncoupling. Conversely, a significant decrease of the mitochondrial KATP+ channel activity was observed. These results suggest a correlation between mitochondrial activities and the manifestation of PCD during the development of somatic embryos. In particular, it is suggested that the KATP+ channel activity could induce an entry of K+ into the matrix, followed by swelling and a release of cytochrome c during proliferation, whereas the alternative pathways, acting as anti-apoptotic factors, may partially counteract PCD events occurring during maturation of somatic embryos.

Activity of a KATP+ channel in Arum spadix mitochondria during thermogenesis

This report demonstrates that mitochondria isolated from thermogenic Arum spadices possess an ATP-sensitive potassium channel--responsible for electrical potential (DeltaPsi) collapse and mitochondrial swelling--whose characteristics are similar to those previously described in pea and wheat mitochondria. In order to study the relationship between this K(ATP)(+) channel and the uncoupled respiration, linked to thermogenesis, K(+) transport activities were compared with those of mitochondria that were isolated from pea stems, soybean suspension cell cultures and Arum tubers. The channel from Arum spadices is highly active and its major features are (i) potassium flux is performed primarily in an inward-rectifying manner; (ii) the influx of K(+) is associated with a matrix volume increase in both energized and non-energized mitochondria; and (iii) its activity depends on the redox state of electron transport chain (ETC) and oxygen availability. In particular, this paper shows that the K(ATP)(+) channel is inwardly activated in parallel with the alternative oxidase (AO). The activation is linked to an ETC-oxidized state and to high oxygen consumption. The putative role of this K(ATP)(+) channel is discussed in relation to flowering of thermogenic Arum spadices.

Isolation of mitochondria from embryogenic cultures of Picea abies (L.) Karst. and Abies cephalonica Loud.: characterization of a K+(ATP) channel.

A valuable method to isolate and purify mitochondria from embryonal masses of two coniferous species (Picea abies [L.] Karst. and Abies cephalonica Loud.) is described. Crude mitochondria from both species were shown to be intact, oxygen consuming (with malate plus glutammate, succinate and NADH as substrates) and well coupled (respiratory control ratio ca. 4). The oxidation of the substrates was only partially KCN-insensitive (alternative oxidase) in some cases. However, these fractions were contaminated by membranes (e.g. plasmalemma, tonoplast, Golgi and endoplasmic reticulum). After purification by a discontinuous Percoll gradient (18, 23, 40%, v/v), three mitochondrial populations were separated. The 0/18 interface fraction was composed mainly of broken and uncoupled mitochondria, while the other two (18/23 and 23/40 interface fractions) contained intact and coupled mitochondria, but only 23/40 interface fraction revealed to be better purified starting from both coniferous embryonal masses. In the latter purified fraction, the presence of a cyclosporin A-sensitive KATP+ channel was demonstrated. These findings were discussed in the light of the potential use of these mitochondrial fractions in bioenergetic studies, or in the involvement of these organelles to stress response in conifers.

Species-specific reversal of stem xylem embolism after a prolonged drought correlates to endpoint concentration of soluble sugars

Recent reports on tree mortality associated with anomalous drought and heat have raised interest into processes underlying tree resistance/resilience to water stress. Hydraulic failure and carbon starvation have been proposed as main causes of tree decline, with recent theories treating water and carbon metabolism as interconnected processes. We subjected young plants of two native (Quercus pubescens [Qp] and Prunus mahaleb [Pm]) and two invasive (Robinia pseudoacacia [Rp] and Ailanthus altissima [Aa]) woody angiosperms to a prolonged drought leading to stomatal closure and xylem embolism, to induce carbon starvation and hydraulic failure. At the end of the treatment, plants were measured for embolism rates and NSC content, and re-irrigated to monitor recovery of xylem hydraulics. Data highlight different hydraulic strategies in native vs invasive species under water stress, and provide physiological explanations for species-specific impacts of recent severe droughts. Drought-sensitive species (Qp and Rp) suffered high embolism rates and were unable to completely refill xylem conduits upon restoration of water availability. Species that better survived recent droughts were able to limit embolism build-up (Pm) or efficiently restored hydraulic functionality after irrigation (Aa). Species-specific capacity to reverse xylem embolism correlated to stem-level concentration of soluble carbohydrates, but not to starch content.

Involvement of apyrase in the regulation of the adenylate pool by adenylate kinase in plant mitochondria

Abstract In this paper, the relationship between adenylate kinase and apyrase activities in controlling the adenylate pool of pea stem mitochondria was examined. Non-purified mitochondria possess an adenylate kinase, localised on the outer surface of the inner membrane, and an apyrase on the surface of the outer membrane. These organelles, energised by succinate, exhibit a cyanide-insensitive ATP synthesis, which is inhibited by P 1 ,P 5 -di(adenosine-5′)pentaphosphate (Ap5A) and carboxyatractylate (CAtr). This synthesis can also be followed either as AMP-induced dissipation of the electrical potential, that is reversed by CAtr and prevented by Ap5A, or as state 4 respiration which is partially inhibited by Ap5A and CAtr. However, when Percoll-purified mitochondria are used, state 4 respiration is insensitive to both inhibitors. Commercial apyrase exogenously supplied to purified mitochondria (which do not have apyrase activity) restores Ap5A- and CAtr-sensitive state 4 oxygen consumption. These results can be interpreted as follows. Apyrase, probably of cytoskeleton or endoplasmic reticulum origin and still adhering to non-purified mitochondria, produces AMP by hydrolysing ADP. AMP, acting as co-substrate with endogenous ATP, is utilised by adenylate kinase (reverse reaction) to generate ADP which, once entered the mitochondrial matrix, is available for ATP synthase.

The Permeability Transition in Plant Mitochondria: The Missing Link

The synthesis of ATP in mitochondria is dependent on a low permeability of the inner membrane. Nevertheless, mitochondria can undergo an increased permeability to solutes, named permeability transition (PT) that is mediated by a permeability transition pore (PTP). PTP opening requires matrix Ca2+ and leads to mitochondrial swelling and release of intramembrane space proteins (e.g., cytochrome c). This feature has been initially observed in mammalian mitochondria and tentatively attributed to some components present either in the outer or inner membrane. Recent works on mammalian mitochondria point to mitochondrial ATP synthase dimers as physical basis for PT, a finding that has been substantiated in yeast and Drosophila mitochondria. In plant mitochondria, swelling and release of proteins have been linked to programmed cell death, but in isolated mitochondria PT has been observed in only a few cases and in plant cell cultures only indirect evidence is available. The possibility that mitochondrial ATP synthase dimers could function as PTP also in plants is discussed here on the basis of the current evidence. Finally, a hypothetical explanation for the origin of PTP is provided in the framework of molecular exaptation.

Water relations of an invasive halophyte (Spartina patens): osmoregulation and ionic effects on xylem hydraulics

Spartina patens (Ait.) Muhl. is a grass native to the Atlantic coastal area of North America currently invading salt marsh ecosystems in several regions of Europe. We investigated leaf water relations and hydraulics, gas exchange, nitrogen and starch content in two populations of S. patens growing under contrasting salinity levels in a salt marsh and in a dune system in order to assess its functional plasticity as a factor contributing to its invasive potential. The analysis of leaf water relations revealed a suite of mechanisms adopted by S. patens to overcome salt and drought stress while maintaining relatively invariant leaf morphological traits and plant biomass. In particular, salt marsh plants experiencing severe water stress underwent greater osmoregulation and leaf hydraulic adjustment than dune plants. We also present the first experimental evidence for salt-mediated regulation of xylem hydraulic efficiency in a halophytic grass and suggest that it is an important functional trait allowing plants growing in saline habitats to cope with a restricted water supply. The functional plasticity of leaf water relations and xylem hydraulics emerges as a key trait underlying the competitive ability and invasive potential of S. patens.