Alberto Bertolini

Plant Flavonoids—Biosynthesis, Transport and Involvement in Stress Responses

This paper aims at analysing the synthesis of flavonoids, their import and export in plant cell compartments, as well as their involvement in the response to stress, with particular reference to grapevine (Vitis vinifera L.). A multidrug and toxic compound extrusion (MATE) as well as ABC transporters have been demonstrated in the tonoplast of grape berry, where they perform a flavonoid transport. The involvement of a glutathione S-transferase (GST) gene has also been inferred. Recently, a putative flavonoid carrier, similar to mammalian bilitranslocase (BTL), has been identified in both grape berry skin and pulp. In skin the pattern of BTL expression increases from véraison to harvest, while in the pulp its expression reaches the maximum at the early ripening stage. Moreover, the presence of BTL in vascular bundles suggests its participation in long distance transport of flavonoids. In addition, the presence of a vesicular trafficking in plants responsible for flavonoid transport is discussed. Finally, the involvement of flavonoids in the response to stress is described.

Transport and accumulation of flavonoids in grapevine (Vitis vinifera L.)

Flavonoids are a group of secondary metabolites widely distributed in plants that represent a huge portion of the soluble phenolics present in grapevine (Vitis vinifera L.). These compounds play different physiological roles and are often involved in protection against biotic and abiotic stress. Even if the flavonoid biosynthetic pathways have been largely characterized, the mechanisms of their transport and accumulation in cell wall and vacuole are still not completely understood. This review analyses the known mechanisms of flavonoid uptake and accumulation in grapevine, with reference to the transport models and membrane carrier proteins described in other plant species. The effect of different environmental factors on flavonoid biosynthesis and transporters is also discussed.

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.

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.

Evidence of Phytotoxicity and Genotoxicity in Hordeum vulgare L. Exposed to CeO2 and TiO2 Nanoparticles

Engineered nanoscale materials (ENMs) are considered emerging contaminants since they are perceived as a potential threat to the environment and the human health. The reactions of living organisms when exposed to metal nanoparticles (NPs) or NPs of different size are not well known. Very few studies on NPs–plant interactions have been published, so far. For this reason there is also great concern regarding the potential NPs impact to food safety. Early genotoxic and phytotoxic effects of cerium oxide NPs (nCeO2) and titanium dioxide NPs (nTiO2) were investigated in seedlings of Hordeum vulgare L. Caryopses were exposed to an aqueous dispersion of nCeO2 and nTiO2 at, respectively 0, 500, 1000, and 2000 mg l-1 for 7 days. Genotoxicity was studied by Randomly Amplified Polymorphism DNA (RAPDs) and mitotic index on root tip cells. Differences between treated and control plants were observed in RAPD banding patterns as well as at the chromosomal level with a reduction of cell divisions. At cellular level we monitored the oxidative stress of treated plants in terms of reactive oxygen species (ROS) generation and ATP content. Again nCeO2 influenced clearly these two physiological parameters, while nTiO2 were ineffective. In particular, the dose 500 mg l-1 showed the highest increase regarding both ROS generation and ATP content; the phenomenon were detectable, at different extent, both at root and shoot level. Total Ce and Ti concentration in seedlings was detected by ICP-OES. TEM EDSX microanalysis demonstrated the presence of aggregates of nCeO2 and nTiO2 within root cells of barley. nCeO2 induced modifications in the chromatin aggregation mode in the nuclei of both root and shoot cells.

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.

Fulvic acid affects proliferation and maturation phases in Abies cephalonica embryogenic cells.

Embryogenic cell masses (ECM) of Abies cephalonica were grown on proliferation media in the presence and absence of fulvic acid (FA), whose molecular composition and conformational rigidity were evaluated by CPMAS-¹³C NMR spectroscopy. To assess the physiological effects of this humic material during proliferation and maturation stages of somatic embryogenesis (SE), proliferation rate, proportion of consecutive developmental stages of pro-embryogenic masses (PEM), cellular ATP and glucose-6-phosphate were evaluated at regular intervals. FA increased the proliferation rate, especially during the early sampling days, and the percentage of PEM in their advanced developmental stage. Cellular ATP and glucose-6-phospahte were increased by FA pre-treatment during the maturation phase. Furthermore, the effects of the anti-auxin p-chlorophenoxyisobutyric acid (PCIB), such as a decrease of growth and the enhancement of PEM III induction, were inverted by FA. Proton pumping ATPase and PPase activities were decreased in microsomes from PCIB-treated ECM, while they increased in the presence of FA. This fulvic matter also induced a delay in somatic embryo formation during the maturation phase. Both the improvement of the PEM proliferation and the reduction of the subsequent maturation process of A. cephalonica are explained by a release from the complex humic structure of low molecular-weight molecules, which may interact with the plant hormonal signaling pathway. These effects appear to be related to the hydrophilic and conformationally labile nature of FA. The structure-activity relationship observed here suggests that the influence of FA on ECM may be attributed to specific bioactive molecules that are preferentially released from the FA loose superstructure.

Identification and localization of the bilitranslocase homologue in white grape berries (Vitis vinifera L.) during ripening

A homologue of the mammalian bilirubin transporter bilitranslocase (BTL) (TCDB 2.A.65.1.1), able to perform an apparent secondary active transport of flavonoids, has previously been found in carnation petals and red grape berries. In the present work, a BTL homologue was also shown in white berries from Vitis vinifera L. cv. Tocai/Friulano, using anti-sequence antibodies specific for rat liver BTL. This transporter, similarly to what found in red grape, was localized in the first layers of the epidermal tissue and in the vascular bundle cells of the mesocarp. In addition, a strong immunochemical reaction was detected in the placental tissue and particularly in peripheral integuments of the seed. The protein was expressed during the last maturation stages in both skin and pulp tissues and exhibited an apparent molecular mass of c. 31 kDa. Furthermore, the transport activity of such a carrier, measured as bromosulphophthalein (BSP) uptake, was detected in berry pulp microsomes, where it was inhibited by specific anti-BTL antibodies. The BTL homologue activity exhibited higher values, for both Km and Vmax, than those found in the red cultivar. Moreover, two non-pigmented flavonoids, such as quercetin (a flavonol) and eriodictyol (a flavanone), inhibited the uptake of BSP in an uncompetitive manner. Such results strengthen the hypothesis that this BTL homologue acts as a carrier involved also in the membrane transport of colourless flavonoids and demonstrate the presence of such a carrier in different organs and tissues.

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.

Changes in ATP, glucose-6-phosphate and NAD(P)H cellular levels during the proliferation and maturation phases of Abies alba Mill. embryogenic cultures

The aim of the present study was to evaluate the adenosine triphospate (ATP), glucose-6-phosphate (glu-6P) and reduced form of nicotinamide adenine dinucleotide phosphate (NAD(P)H) cellular levels during the proliferation and maturation phases of Abies alba Mill. somatic embryos. For a better understanding of the dynamics of these parameters during the proliferation cycle, four embryonic cell lines were tested. During the maturation period, three independent experiments were conducted, focused on the effects of PEG-4000 (5 or 10% (w/v)) and abscisic acid (16, 32 or 64 μM) applied together (Experiments A and B) or with addition of gibberellic acid (Experiment C) on the dynamics of bio-energetic molecules and on the mean number of cotyledonary somatic embryos. Our results demonstrated that the cellular levels of bio-energetic molecules strongly depended on the composition of maturation media. Generally, the higher the number of cotyledonary embryos produced, the higher the level of ATP observed after a 2-week maturation period. The cellular level of ATP, glu-6P and NAD(P)H increased, particularly after the transition from the proliferation to the maturation phase when the differentiation and growth of somatic embryos occurred.