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Seed storage in polyethylene bags of a recalcitrant species (Quercus ilex L.): analysis of some bio-energetic and oxidative parameters.
No full textInvolvement of plasma membrane peroxidases and oxylipin pathway in the recovery from phytoplasma disease in apple (Malus domestica)
No full textApple trees (Malus domestica) may be affected by apple proliferation (AP), caused by “Candidatus Phytoplasma mali”. Some plants can spontaneously recover from the disease, which implies the disappearance of symptoms through a phenomenon known as recovery. In this paper it is shown that NAD(P)H peroxidases of leaf plasma membrane-enriched fractions exhibited a higher activity in samples from both AP-diseased and recovered plants. In addition, an increase in endogenous SA was characteristic of the symptomatic plants, since its content increased in samples obtained from diseased apple trees. In agreement, phenylalanine ammonia lyase (PAL) activity, a key enzyme of the phenylpropanoid pathway, was increased too. Jasmonic acid (JA) increased only during recovery, in a phase subsequent to the pathological state, and in concomitance to a decline of salicylic acid (SA). Oxylipin pathway, responsible for JA synthesis, was not induced during the development of AP-disease, but it appeared to be stimulated when the recovery occurred. Accordingly, lipoxygenase (LOX) activity, detected in plasma membrane-enriched fractions, showed an increase in apple leaves obtained from recovered plants. This enhancement was paralleled by an increase of hydroperoxide lyase (HPL) activity, detected in leaf microsomes, albeit the latter enzyme was activated in either the disease or recovery conditions. Hence, a reciprocal antagonism between SA- and JA-pathways could be suggested as an effective mechanism by which apple plants react to phytoplasma invasions, thereby providing a suitable defense response leading to the establishment of the recovery phenomenon
Mitochondrial ferritin distribution among plant organs and its involvement in ascorbate-mediated iron uptake and release
No full textIron sequestration inside the plant cell may be accomplished by ferritins, a class of proteins able to buffer and store this metal in a safe and soluble form. This protein has been shown in plastids and only recently in plant mitochondria. In this paper, we examined the localization of mitochondrial ferritin in different plant organs and its possible involvement in iron uptake and/or release. Ferritin was found only in mitochondria from young organs, such as roots, etiolated stems and, although to a lesser extent, developing leaves. Its presence was not influenced by iron in the hydroponic medium of culture, as well as by the induction of senescence through ethylene treatment. Lipoperoxidation, evaluated as formation of thiobarbituric acid reactive substances (TBARS), was determined in mitochondria isolated from etiolated stems and leaves of plants incubated with FeSO4/citrate and ascorbate. The phenomenon was lower in young stem mitochondria, where ferritin was detected, than in those from leaves, where the protein was undetectable. Therefore, these results indicate that the mitochondrial ferritin was able to sequestrate iron within its shell. A significant increase of TBARS formation was also found in etiolated stem mitochondria incubated, in the absence of supplied iron, with 5 mM ascorbate, which was paralleled by a decrease of iron in mitochondrial ferritin, detected by Inductively Coupled Plasma Mass Spectrum (ICP-MS) measurements after immunoprecipitation. These results indicate that plant mitochondrial ferritin plays a key role in iron buffering through the uptake and release of iron that could be modulated by ascorbate
Attività di perossidasi di superficie e via delle ossilipine in piante di melo soggette a recovery da fitoplasmosi
No full textApple trees (Malus domestica Borkh) may be affected by apple proliferation (AP), caused by ‘Candidatus Phytoplasma mali’. Some plants can spontaneously recover from the disease, which implies the disappearance of symptoms through a phenomenon known as recovery. Recovered apple trees show the disappearance of the symptoms and pathogens from the canopy, but not from the infected roots, where phytoplasmas persist viable and infectious. Recovery of apple plants is linked to an overproduction of hydrogen peroxide in the phloem tissues. The high levels of hydrogen peroxide in the canopy of recovered apple plants could have a direct antimicrobial effect on pathogens and/or a signalling function, in order to activate defence responses triggered by the phytormones jasmonate and salicylate. In general, it can be stated that salicylate promotes resistance against biotrophic pathogens, whereas the jasmonate pathway induces resistance against necrotrophic pathogens and herbivorous insects. Apple plants, infected by AP-disease, initially show an induced mechanism of defence mediated by salicylate but, subsequently, during the recovery, an increase of jasmonate, sinthesized by the oxylipin pathway, occurs
Mitochondrial ferritin distribution in plant organs and its involvement in oxidative stress prevention
No full textFerritins are proteins able to store iron in a safe and soluble form (l). They are localized in the cytoplasm of mammalian cells whereas have a plastidial localization in the plant ones. Only recently this protein has been reported to be present in both animal (2) and plant (3) mitochondria. In this work, we initially examined the distribution of mitochondrial ferritin (MF) in different plant organs of pea. The plants were grown in hydroponic culture, in the dark, for 7 days and then up to 18 days under light-dark (16-8 h) cycles. The plants were grown either in the presence or in the absence of iron (supplied as Fe(SO4)2-citrate: 100 uM/500 uM). Purified mitochondria were isolated from roots (R), etiolated stems (S), and young (YL) and mature leaves (L). The MF was detected after SDS-PAGE and Western blotting using a polyclonal antibody raised against the pea seed ferritin. Figure 1 shows that the MF was detected in young roots and stems, grown without light, and, although to a lesser extent, in young leaves. In contrast, the MF was not detected in mature organs of plants grown in the presence of light even if iron was supplied. In order to verify the involvement of MF in oxidative stress prevention, we also examined the induction of lipoperoxidation, evaluated as malondialdehyde (MDA) formation, in purified mitochondria isolated from both young pea stems and mature leaves. Lipoperoxidation was induced by 10 uM iron in the presence of 200 uM ascorbate. Figure 2a shows that the level of lipoperoxidation was higher in mitochondria isolated from leaves that do not possess ferritin, than mitochondria isolated from etiolated stems. This decrease indicates that iron is taken up by the MF. Lipoperoxidation was also detected in purified mitochondria isolated from etiolated pea stems in the presence of either 200 uM or 5 mM ascorbate, but in the absence of iron. The increase of lipoperoxidation, when mitochondria were incubated with 5 mM ascorbate, indicates a release of iron from the MF (Figure 2b). These results confirm that MF plays a role in the prevention of oxidative stress in plant mitochondria, particularly in the initial stages of organ development. Therefore, MF is involved in the metabolism of iron by its release and/or acquisition that seems to be finely regulated by ascorbate concentration
Characterization of flavonoid accumulation in cell suspension cultures of Vitis vinifera L. cv. Limberger
No full textLIPOXYGENASE ACTIVITY AND HYDROPEROXIDE FORMATION IN COFFEE (Coffea arabica L.) CHERRIES CULTIVATED BY DIFFERENT AGRONOMIC TECHNIQUES
Full text linkIt is widely accepted that biotic stress induces acidic lipoxygenase (LOX) activity mainly due to the effects of methyl jasmonate [1]. The activation of LOX pathway leads to the production of a large amount of oxidation products, which may affect the coffee organoleptic profile [2]. Hitherto the presence of LOX in coffee (Coffea arabica L.) plants is still scarcely examined [3], therefore the aim of this study was to investigate the possible involvement of this enzyme in oxidative stress of cherries from plants cultivated in organic and conventional manners.
Primary and secondary oxidation products were evaluated in three separate cherry fractions: pulp, parchment seed and green coffee. Hydroperoxides, measured by HPLC, were highest in parchment seed and lowest in green coffee, but no significant difference was found between the samples obtained from the two cultivations. However, the pulp fraction from organic cultivated plants had a larger amount of primary peroxidation products, when compared to that from conventionally cultivated plants. The presence of LOX was then examined in all cherry fractions by immunochemical analysis. Only the pulp fraction showed an 84 kDa protein that exhibited a cross-reactivity against a pea LOX antibody. Subsequently, the presence of LOX was confirmed in heavy (28000 g) and light (100000 g) membrane pulp fractions by an enzymatic assay, immunochemical method and hydroperoxide formation. In both organic and conventional cultivation systems, the light membrane showed the highest LOX activity, measured as conjugated diene formation, at pH 8.0. Instead, the heavy membrane fraction highlighted a different pH optimum, which was found to be 5.5 in pulps from organic cultivations and 8.0 in the conventional ones, respectively. All the activities reported were inhibited by nordihydroguaiaretic acid (NDGA) and caffeic acid, well known LOX inhibitors. The NDGA-sensitive hydroperoxide production was simultaneously confirmed by RP-HPLC analysis. Oxidative stress was also detected by evaluating the antioxidant activity in all fractions. A lipophilic extraction, performed using dioxane:water (95:5, v/v), revealed that parchment seeds possessed the lowest antioxidant capacity. On the other hand, a significant difference in the antioxidative capacity was detected in parchment seeds from organic and conventionally cultivated plants.
Taken together, these results show, for the first time, the presence of LOX in coffee cherries, whose activity was associated to membrane fractions. Furthermore, the cultivation techniques seem to induce the activation of different isoenzymes. In particular, the organic ones were subjected to an oxidative stress in coffee fruits leading to the expression of an acidic LOX activity, detectable in the pulp fraction.
[1] Buzi A., et al. (2004). J. Phytopathol., 152: 34-42.
[2] Kohlmann M., et al. (1999) Eur. J. Biochem. 260:885-895.
[3] Rojas M.L., et al. (1993). Physiol. Mol. Plant Path., 43: 209-219
Stato energetico e capacità antiossidante in semi recalcitranti di leccio (Quercus ilex L.) conservati in sacchetti di polietilene.
No full textIl leccio (Quercus ilex L.) costituisce una tipica specie recalcitrante dell’area mediterranea che si caratterizza per una debole dormienza e per una spiccata sensibilità alla disidratazione nei semi. Per descrivere e comprendere meglio i meccanismi fisiologici e biochimici che sottostanno a queste peculiarità, sono stati misurati nel corso della conservazione alcuni parametri tecnologici e biologici. I semi, mantenuti in condizioni di elevata umidità e bassa temperatura, sono stati conservati secondo le usuali metodiche in bidoni riempiti con torba umida per un intero anno. Tale modalità è stata confrontata con una soluzione che prevedeva l’uso di sacchetti in polietilene. L’accorgimento adottato ha consentito di mantenere un livello accettabile di germinazione per un periodo superiore di circa 3 mesi rispetto al controllo e di rallentare il deterioramento del seme grazie a un miglior controllo dei danni provocati dal metabolismo ossidativo. La barriera costituita dal film plastico ha infatti limitato gli scambi gassosi, prevenendo le alterazioni dovute a una respirazione troppo elevata e alla liberazione di H2O2. L’ambiente ipossico ha mantenuto un miglior equilibrio ossidoriduttivo, preservando il contenuto di glutatione ridotto e di ATP. La conservazione nei sacchetti ha inoltre favorito l’accumulo di etilene nell’atmosfera di stoccaggio e ha abbassato le attività metaboliche dei semi, nonostante il loro intrinseco stato di debole dormienza. Un’appropriata modulazione di questi fattori biologici potrebbe consentire il prolungamento del periodo di conservazione in semi recalcitranti
Lipoxygenase and hydroperoxide lyase activities in olive pulps from Ghiacciolo and Nostrana cultivars
No full textBiochemical and immunochemical similarities among mammalian bilitranslocase and a plant flavonoid translocator
Full text linkFlavonoids are a large class of plant secondary metabolites,
belonging to polyphenol family, which possess pharmacological and nutritional
properties. Their synthesis takes place only in plants, while mammals can
acquire them only with diet. It has been demonstrated that flavonoid uptake
occurs in rat also by the activity of bilitranslocase, a carrier that is involved
in anion transport in liver cell, vascular endothelium and gastric mucosa. A
sequence of bilitranslocase interacting with flavonoid moieties is already known
and characterized. Antibody raised against such protein epitope were shown
to exhibit cross-reactivity against plant membrane proteins in tissues involved
in flavonoid transport and accumulation, such as teguments of carnation
petals and skin of grape berries. Further immunolocalization studies allowed
to demonstrate the presence of cross-reacting protein not only at the level of
tegumental tissues, but also associated to sieve elements and seed teguments
in grape berries
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