1,721,047 research outputs found
Spermine Regulates Pollen Tube Growth by Modulating Ca2+-Dependent Actin Organization and Cell Wall Structure
Full text linkProper growth of the pollen tube depends on an elaborate mechanism that integrates several molecular and cytological sub-processes and ensures a cell shape adapted to the transport of gametes. This growth mechanism is controlled by several molecules among which cytoplasmic and apoplastic polyamines. Spermine (Spm) has been correlated with various physiological processes in pollen, including structuring of the cell wall and modulation of protein (mainly cytoskeletal) assembly. In this work, the effects of Spm on the growth of pear pollen tubes were analyzed. When exogenous Spm (100 μM) was supplied to germinating pollen, it temporarily blocked tube growth, followed by the induction of apical swelling. This reshaping of the pollen tube was maintained also after growth recovery, leading to a 30–40% increase of tube diameter. Apical swelling was also accompanied by a transient increase in cytosolic calcium concentration and alteration of pH values, which were the likely cause for major reorganization of actin filaments and cytoplasmic organelle movement. Morphological alterations of the apical and subapical region also involved changes in the deposition of pectin, cellulose, and callose in the cell wall. Thus, results point to the involvement of Spm in cell wall construction as well as cytoskeleton organization during pear pollen tube growth
Spermine either delays or promotes cell death in Nicotiana tabacum L. corolla depending on the floral developmental stage and affects the distribution of transglutaminase
Full text linkThe role of spermine (SM) was studied to verify if SM supplied to Nicotiana tabacum flower can modulate programmed cell death (PCD) of the corolla. SM has strong effects on the development and senescence of excised flowers despite its low physiological levels. The timing and duration of SM treatment is a key factor; SM counteracts PCD (verified by morphological observations, pigment contents and DNA laddering) only in the narrow developmental window of corolla expansion. Before and after, SM promotes PCD. SM exerts its pro-survival role by delaying fresh weight loss, by inhibiting reduction of pigments and finally by preventing DNA degradation. Moreover, SM deeply alters the distribution of the PA-conjugating enzyme transglutaminase (TGase). TGase is present in the epidermis during development, but it sprays also in the cell walls of inner parenchyma at senescence. After SM treatment, parenchyma cells accumulate TGase, increase in size and their cell walls do not undergo stiffening contrarily to control cells. The subcellular localization of TGase has been validated by biolistic-transformation of onion epidermal cells.
Results indicated that SM is a critical factor in the senescence of N. tabacum corolla by controlling biochemical and morphological parameters; the lasts are probably interconnected with the action of TGase
Polyamines in pollen: from microsporogenesis to fertilization
Full text linkThe entire pollen life span is driven by polyamine (PA) homeostasis, achieved through fine regulation of their biosynthesis, oxidation, conjugation, compartmentalization, uptake, and release. The critical role of PAs, from microsporogenesis to pollen–pistil interaction during fertilization, is suggested by high and dynamic transcript levels of PA biosynthetic genes, as well as by the activities of the corresponding enzymes. Moreover, exogenous supply of PAs strongly affects pollen maturation and pollen tube elongation. A reduction of endogenous free PAs impacts pollen viability both in the early stages of pollen development and during fertilization. A number of studies have demonstrated that PAs largely function by modulating transcription, by structuring pollen cell wall, by modulating protein (mainly cytoskeletal) assembly as well as by modulating the level of reactive oxygen species. Both free low-molecular weight aliphatic PAs, and PAs conjugated to proteins and hydroxycinnamic acids take part in these complex processes. Here, we review both historical and recent evidence regarding molecular events underlying the role of PAs during pollen development . In the concluding remarks, the outstanding issues and directions for future research that will further clarify our understanding of PA involvement during pollen life are outlined
Distribution of Transglutaminase in Pear Pollen Tubes in Relation to Cytoskeleton and Membrane Dynamics
Full text linkTransglutaminases (TGases) are ubiquitous enzymes that take part in a variety of cellular functions. In the pollen tube, cytoplasmic TGases are likely to be involved in the incorporation of primary amines at selected peptide-bound glutamine residues of cytosolic proteins (including actin and tubulin), while cell wall-associated TGases are believed to regulate pollen tube growth. Using immunological probes, we identified TGases associated with different subcellular compartments (cytosol, membranes, and cell walls). Binding of cytosolic TGase to actin filaments was shown to be Ca2+-dependent. The membrane TGase is likely associated with both Golgi-derived structures and the plasma membrane, suggesting a Golgi-based exocytotic delivery of TGase. Association of TGase with the plasma membrane was also confirmed by immunogold transmission electron microscopy. Immunolocalization of TGase indicated that the enzyme was present in the growing region of pollen tubes, and that the enzyme co-localizes with cell wall markers. Bidimensional electrophoresis indicated that different TGase isoforms were present in distinct subcellular compartments, suggesting either different roles or different regulatory mechanisms of enzyme activity. The application of specific inhibitors showed that the distribution of TGase in different subcellular compartments was regulated by both membrane dynamics and cytoskeleton integrity, suggesting that delivery of TGase to the cell wall requires the transport of membranes along cytoskeleton filaments. Taken together, these data indicate that a cytoplasmic TGase interacts with the cytoskeleton, while a different TGase isoform, probably delivered via a membrane/cytoskeleton-based transport system, is secreted in the cell wall of pear pollen tubes where it might play a role in the regulation of apical growth
Transgenic rice as a vehicle for the production of the industrial enzyme transglutaminase
No full textPollen Transglutaminase in Pear Self Incompatibility and Relationships with S-RNases and S-Allele Variability.
No full textThe pear self-incompatibility in the cv Abbé Fètel (P. communis) has been investigated in order to identify the S-alleles and pollen putative determinant transglutaminase (TGase), an enzyme able to post-translationally forming bridges among proteins. In the self-pollinated style (incompatible system), the activity of TGase is stimulated in comparison to style-pollinated with compatible pollen (compatible system), and high molecular mass cross-linked products are formed. High mass aggregates of tubulin, and punctuate aggregates of actin were observed in the incompatible system, suggesting a role of cytoskeleton in SI. In vitro experiments with purified pollen TGase and purified actin and tubulin, also incubated with kinesin and myosin motor-proteins, show that the inhibition of tube growth in incompatible crossing might be mediated by a cytoskeleton abnormal reorganisation caused by cross-linked protein networks catalysed by TGase. A molecular approach to clone pear TGase and S-RNases has been performed. The high homology between the pear and apple TGase sequences suggests that the good knowledge of the apple isoform can be transferred to pear. Contrarily to the pollen determinant, S- RNases have been very well characterised in pear and several S-alleles were identified. The authors have also approached the possible key role carried out by TGase in controlling the interaction between stylar RNases and pollen determinants of the SI mechanism. The update characterization of the European pear S-allele variability allows to set the pear interfertility groups
Influence of climate changes on cross allergies: possible involvement of pollen transglutaminase
No full textAllergies are a complex of symptoms derived from altered IgE-mediated reactions of the immune system towards substances known as allergens. Allergic sensibilization can be of food or respiratory origin and, in particular, apple and hazelnut allergens have been identified in pollens or fruits. Allergic cross-reactivity can occur in a patient reacting to similar allergens from different origins, justifying the research in both systems as in Europe a greater number of people suffers from apple fruit allergy, but little evidence exists about pollen. Apple fruit allergies are due to four different classes of allergens (Mal d 1, 2, 3, 4), whose allergenicity is related both to genotype and tissue specificity; therefore I have investigated their presence also in pollen at different time of germination to clarify the apple pollen allergenic potential.
I have observed that the same four classes of allergens found in fruit are expressed at different levels also in pollen, and their presence might support that the apple pollen can be considered allergenic as the fruit, deducing that apple allergy could also be indirectly caused by sensitization to pollen.
Climate changes resulting from increases in temperature and air pollution influence pollen allergenicity, responsible for the dramatic raise in respiratory allergies (hay fever, bronchial asthma, conjunctivitis). Although the link between climate change and pollen allergenicity is proven, the underlying mechanism is little understood.
Transglutaminases (TGases), a class of enzymes able to post-translationally modify proteins, are activated under stress and involved in some inflammatory responses, enhancing the activity of pro-inflammatory phospholipase A2, suggesting a role in allergies. Recently, a calcium-dependent TGase activity has been identified in the pollen cell wall, raising the possibility that pollen TGase may have a role in the modification of pollen allergens reported above, thus stabilizing them against proteases. This enzyme can be involved also in the transamidation of proteins present in the human mucosa interacting with surface pollen or, finally, the enzyme itself can represent an allergen, as suggested by studies on celiac desease. I have hypothesized that this pollen enzyme can be affected by climate changes and be involved in exhacerbating allergy response. The data presented in this thesis represent a scientific basis for future development of studies devoted to verify the hypothesis set out here.
First, I have demonstrated the presence of an extracellular TGase on the surface of the grain observed either at the apical or the proximal parts of the pollen-tube by laser confocal microscopy (Iorio et al., 2008), that plays an essential role in apple pollen-tube growth, as suggested by the arrest of tube elongation by TGase inhibitors, such as EGTA or R281. Its involvement in pollen tube growth is mainly confirmed by the data of activity and gene expression, because TGase showed a peak between 15 min and 30 min of germination, when this process is well established, and an optimal pH around 6.5, which is close to that recorded for the germination medium. Moreover, data show that pollen TGase can be a glycoprotein as the glycosylation profile is linked both with the activation of the enzyme and with its localization at the pollen cell wall during germination, because from the data presented seems that the active form of TGase involved in pollen tube growth and pollen-stylar interaction is more exposed and more weakly bound to the cell wall.
Interestingly, TGase interacts with fibronectin (FN), a putative SAMs or psECM component, inducing possibly intracellular signal transduction during the interaction between pollen-stylar occuring in the germination process, since a protein immunorecognised by anti-FN antibody is also present in pollen, in particular at the level of pollen grain cell wall in a punctuate pattern, but also along the shank of the pollen tube wall, in a similar pattern that recalls the signal obtained with the antibody anti TGase. FN represents a good substrate for the enzyme activity, better than DMC usually used as standard substrate for animal TGase. Thus, this pollen enzyme, necessary for its germination, is exposed on the pollen surface and consequently can easily interact with mucosal proteins, as it has been found germinated pollen in studies conducted on human mucus (Forlani, personal communication).
I have obtained data that TGase activity increases in a very remarkable way when pollen is exposed to stressful conditions, such as climate changes and environmental pollution. I have used two different species of pollen, an aero allergenic (hazelnut, Corylus avellana) pollen, whose allergenicity is well documented, and an enthomophylus (apple, Malus domestica) pollen, which is not yet well characterized, to compare data on their mechanism of action in response to stressors. The two pollens have been exposed to climate changes (different temperatures, relative humidity (rH), acid rain at pH 5.6 and copper pollution (3.10 µg/l)) and showed an increase in pollen surface TGase activity that is not accompanied to an induced expression of TGase immunoreactive protein with AtPNG1p. Probably, climate change induce an alteration or damage to pollen cell wall that carries the pollen grains to release their content in the medium including TGase enzyme, that can be free to carry out its function as confirmed by the immunolocalisation and by the in situ TGase activity assay data; morphological examination indicated pollen damage, viability significantly reduced and in acid rain conditions an early germination of apple pollen, thus possibly enhancing the TGase exposure on pollen surface.
Several pollen proteins were post-translationally modified, as well as mammalian sPLA2 especially with Corylus pollen, which results in its activation, potentially altering pollen allergenicity and inflammation. Pollen TGase activity mimicked the behaviour of gpl TGase and AtPNG1p in the stimulation of sPLA2, even if the regulatory mechanism seems different to gpl TGase, because pollen TGase favours an intermolecular cross-linking between various molecules of sPLA2, giving rise to high-molecular protein networks normally more stable. In general, pollens exhibited a significant endogenous phospholipase activity and it has been observed differences according to the allergenic (Corylus) or not-well characterized allergenic (Malus) attitude of the pollen. However, even if with a different intensity level in activation, pollen enzyme share the ability to activate the sPLA2, thus suggesting an important regulatory role for the activation of a key enzyme of the inflammatory response, among which my interest was addressed to pollen allergy.
In conclusion, from all the data presented, mainly presence of allergens, presence of an extracellular TGase, increasing in its activity following exposure to environmental pollution and PLA2 activation, I can conclude that also Malus pollen can behave as potentially allergenic. The mechanisms described here that could affect the allergenicity of pollen, maybe could be the same occurring in fruit, paving the way for future studies in the identification of hyper- and hypo- allergenic cultivars, in preventing environmental stressor effects and, possibly, in the production of transgenic plants
PROGETTO STRATEGICO D’ATENEO 2006 Titolo: CROSS ALLERGENS IN POLLEN AND FRUITS: MODULATION BY CLIMATE CHANGES OF THEIR ALLERGENIC POTENTIAL
No full textRIASSUNTO
Stato dell’arte. Le allergie, l’epidemia del XXI secolo, sono in costante aumento nei paesi Occidentali ma i fattori che contribuiscono a tale incremento non sono ancora noti. Allergeni di piante di famiglie diverse, aventi un rilevante impatto epidemiologico e diverso potenziale allergenico a seconda delle aree geografiche, sono coinvolti nelle allergie da polline e alimentari (i.e. Graminaceae, Betulaceae, Corylaceae, Rosaceae); anche una cross-reattività tra allergeni di differenti specie è stata ben documentata (es. betulla/melo). La frequenza e la gravità delle reazioni allergiche sono correlate alla via di sensibilizzazione (contatto, inalazione, ingestione) e alla stabilità degli allergeni. Inoltre, i cambiamenti climatici e l’inquinamento sembrano aggravare le allergie, agendo direttamente sulle difese immunitarie degli individui e provocando una risposta allergica mediata da IgE, oppure causando modificazioni degli allergeni stessi incrementandone la capacità allergenica, con evidenti aumenti dei costi in termini di terapia e di capacità lavorativa (D’Amato et al., 2002). Mentre sono presenti numerosi studi sulla relazione fra inquinanti e risposta immunitaria (Beggs, 2004; D’Amato, 2000), sono pochi e insufficienti i dati relativi alla modulazione/ modificazione degli allergeni dovuta a fattori di stress. È risaputo che le transglutaminasi (TGasi), una classe di enzimi capaci di modificare post-traduzionalmente le proteine (Griffin et al., 2002), aumentano considerevolmente l’attività enzimatica in condizioni di stress e sono coinvolte in alcune risposte infiammatorie, suggerendone un ruolo anche nelle allergie, come già riportato per la congiuntivite allergica (Sohn et al., 2003). Le TGasi possono modificare direttamente gli antigeni pollinici o attivare la fosfolipasi A (sPLA2), responsabile delle risposte infiammatorie dopo contatto diretto con polline.
Obiettivi complessivi del progetto. a) migliorare con approcci multidisciplinari le conoscenze di base sugli allergeni, sulla ‘cross allergenicità’ e sui meccanismi molecolari coinvolti nell’aumento dell’allergenicità in relazione alle modificazioni ambientali; b) verificare la correlazione ambiente-allergia attraverso la comparazione di dati clinici e ambientali in zone a diverso inquinamento; c) verificare se i principali allergeni di melo siano substrati per le TGasi; d) convalidare in nocciolo (Corylus avellana), un’altra specie la cui cross-allergenicità polline/frutto è ben documentata, i risultati ottenuti in melo.
Obiettivi a breve-medio termine dello start up. a) individuare con strumenti bioinformatici motivi comuni negli allergeni del polline e frutto; b) caratterizzare la variabilità genetica di melo in relazione alla cross-allergenicità polline/frutto in collezioni di germoplasma; c) valutare l’attività TGasica su substrati allergenici e sPLA2 in vitro; d) verificare i meccanismi molecolari con cui i diversi agenti stressanti, compreso l’inquinamento, possono influenzare la vitalità e le attività enzimatiche del polline; e) valutare la correlazione tra composizione pollinica e inquinanti ambientali sulle patologie allergiche in bambini allergici provenienti da aree urbane e rurali.
Ampliamento delle conoscenze e innovazione a medio-lungo termine. a) identificare le sequenze dei principali allergeni associate a diversi gradi di allergenicità, al fine di produrre cultivar ipoallergeniche con evidenti riflessi nutrizionali e di prevenzione; b) valutare i meccanismi molecolari di azione delle TGasi vegetali nell’influenzare l’allergenicità anche in relazione a variazioni climatiche; c) analizzare l’evoluzione delle allergie in relazione ai cambiamenti climatici raccogliendo dati da 4 anni di “follow up” su bambini allergici per l’attuazione di strategie preventive e terapeutiche. La scelta del “follow up” sui bambini è giustificata dal fatto che le allergie sono molto aumentate a carico di questi pazienti e che questi sono più recettivi ..
Proteine modificate post-traduzionalmente da transglutaminasi durante la morte cellulare programmata
No full textLe conoscenze sulla PCD nelle piante sono molto meno avanzate di quelle acquisite negli organismi animali. Frequentemente, la morte programmata nelle piante rappresenta lo stadio terminale del differenziamento. Nel caso dei fiori la fecondazione può determinare la senescenza e l’abscissione dei petali oppure questi eventi possono essere programmati indipendentemente.
Uno dei fattori rilevanti nell’apoptosi delle cellule animali è risultata essere la modificazione post-traduzionale delle proteine e in particolare quella catalizzata dalle transglutaminasi, la cui presenza e l’attività sono state riscontrate in diversi tessuti in apoptosi. L’enzima catalizza la coniugazione di lisina o ammine primarie a glutamine. Con le poliamine si possono formare derivati mono- e bis –sostituiti dando origine a ponti inter- od intra-proteici a significato strutturale. La conformazione dei substrati può esserne modificata e si possono formare generate reti sopramolecolari. Le poliamine alifatiche libere hanno un ruolo ben stabilito nella stimolazione della divisione cellulare, dello sviluppo e nel dilazionare la senescenza
Si intende determinare quali siano le proteine, coinvolte nei fenomeni di morte cellulare programmata, modificate post-traduzionalmente, applicando nuove tecnologie informatiche e morfo-funzionali. Si studieranno gli effetti di tali modificazioni sui metabolismi cellulari
La ricerca è volta a determinare il ruolo della transglutaminasi nelle cellule dei petali di Nicotiana durante la senescenza e la PCD. A tal fine applicando approcci di tipo bio-informatico e morfo-funzionale si intende approfondire l’analisi sia degli enzimi che dei loro substrati proteici. Gli obbiettivi sono quelli di: (1) purificare la transglutaminasi vegetale; (II) microsequenziare l’enzima; (III) produrre un modello strutturale della proteina e compararlo con quello di TGasi note; (IV) comparare le funzioni dell’enzima vegetale con la TGasi di Nematode, con cui vi sono indizi che condivida sequenze omologhe, e con quelle di mammifero con cui sembra invece che non ci siano omologie di sequenza; (V) valutare se l’enzima ha attività deamidasica; (VI e VII) studiare la localizzazione, in relazione a senescenza e PCD, dell’enzima e dei substrati e studiare le modificazioni di proteine: (VIII) citoscheletriche, (IX) di parete (X) plastidiali. Un altro obbiettivo con scopi applicativi consiste nel comprendere il meccanismo molecolare di “ringiovanimento” della spermina su fiori recisi o altri prodotti freschi
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