Anna Maria Bregoli

Jasmonate-induced transcriptional changes suggest a negative interference with the ripening syndrome in peach fruit

Peach (Prunus persica L. Batsch) was chosen as a model to shed light on the physiological role of jasmonates (JAs) during fruit ripening. To this aim, the effects of methyl jasmonate (MJ, 0.40 mM) and propyl dihydrojasmonate (PDJ, 0.22 mM), applied in planta at different fruit developmental stages, on the time-course of ethylene production and fruit quality traits were evaluated. MJ-induced changes in fruit transcriptome at harvest and the expression profiling of relevant JA-responsive genes were analysed in control and JA-treated fruit. Exogenously applied JAs affected the onset of ripening depending upon the fruit developmental stage, with PDJ being more active than MJ. Both compounds enhanced the transcription of allene oxide synthase (PpAOS1), the first specific enzyme in the biosynthesis of jasmonic acid, and altered the pattern of jasmonic acid accumulation. Microarray transcriptome profiling showed that MJ down-regulated some ripening-related genes, such as 1-aminocyclopropane-1-carboxylic acid oxidase (PpACO1) and polygalacturonase (PG), and the transcriptional modulator IAA7. MJ also altered the expression of cell wall-related genes, namely pectate lyase (PL) and expansins (EXPs), and up-regulated several stress-related genes, including some of those involved in JA biosynthesis. Time-course expression profiles of PpACO1, PL, PG, PpExp1, and the transcription factor LIM confirmed the array results. Thus, in peach fruit, exogenous JAs led to a ripening delay due to an interference with ripening- and stress/defence-related genes, as reflected in the transcriptome of treated fruit at harvest.

Transglutaminase activity during senescence and programmed cell death in the corolla of tobacco ( Nicotiana tabacum ) flowers

Corolla life span of undetached flowers of Nicotiana tabacum was divided into stages from the closed corolla (stage 1) through anthesis (stage 5) to death (stage 9). Senescence began around stage 6 in the proximal part, concomitantly with DNA laddering. Nuclear blebbing, DNA laddering, cell wall modification, decline in protein, water, pigment content and membrane integrity were observed during senescence and PCD. Transglutaminase activity was measured as mono- and bis-derivatives of putrescine (mono-PU; bis-PU) and bis-derivatives of spermidine (bis-SD). Bis-derivatives decreased with the progression of senescence, while mono-PU increased during early senescence; derivatives were present in different amounts in the proximal and distal parts of the corolla. In excised flowers, exogenous spermine delayed senescence and PCD, and caused an increase in free and acid-soluble conjugated PA levels. Bis-PU was the most abundant PA-derivative before DNA laddering stage; thereafter, bis-PU generally decreased and mono-PU became the most abundant derivative.

Transglutaminase-catalyzed modification of cytoskeletal proteins by polyamines during the germination of Malus domestica pollen

Abstract We investigated polyamine linkage to different structural proteins in pollen of Malus domestica Borkh. cv Red Chief at different phases of germination. This linkage has the characteristics of covalent linkages, indeed, it could be catalyzed by transglutaminase (TGase; EC 2.3.2.13). This assumption is supported by: (1) formation of a labelled TCA pellet and selective labelling of endogenous proteins by covalent binding of radioactive polyamines and (2) cross-reactivity of two different polyclonal antibodies against mammalian TGases; western blot analysis allowed us to detect a protein of about 80 kDa in both rehydrated ungerminated and germinated pollen. TGase activity was high at 90 min after germination and was influenced by Ca2+ supply only in the rehydrated ungerminated pollen. Extraction by Triton X-100 suggests that pollen TGase was at least partially membrane-bound. The enzyme catalyzed the incorporation of polyamines mainly into proteins having a molecular mass of 43 kDa and 52–58 kDa in both ungerminated and germinated pollen. These bands matched immunolabelled spots identified by mouse monoclonal anti-actin and anti-α-tubulin antibodies. Supplying exogenous actin and tubulin in a cell-free extract of rehydrated ungerminated and germinated pollen enhanced the activity. Autoradiography of the SDS-PAGE of these samples clearly showed that both actin and tubulin were substrates of TGase. Thus, the pollen TGase may be involved in the rapid cytoskeletal rearrangement which takes place during rehydration of ungerminated pollen and organization and growth of pollen tubes.

Acclimation of chloroplast transglutaminase to high NaClconcentration in a polyamine-deficient variant strain of Dunaliella salina and in its wild type

Summary The wild type (Wt) and the polyamine-deficient strain (PA − vs) of the halotolerant Dunaliella salina were subjected to stress caused by 3.5 mol/L NaCl concentration. The chloroplasts were isolated and the molecular aspects of their reaction to salt stress were studied together with their recovery response to these hyper-saline conditions. In the Wt, the photosynthetic complexes were found to be severely affected by salt stress under light conditions. Transglutaminases, which are present in chloroplasts as two units of 25 and 50 kDa, were immunorecognized by antibodies raised against rat prostatic gland transglutaminase. The amount, in particular that of the 50 kDa unit, underwent an immediate change following hyper-saline stress. These concentration changes were found to coincide with variations in enzymic activity, which is also affected by the presence or absence of light. The PA − vs has a concentration of proteins and chlorophylls which is much lower than that of the Wt. In addition, the PA − vs appeared to be more severely affected by both salt and subculture stresses. Its recovery time was also longer. Its TGase activity increased after salt stress and was always higher in the light than in the dark, except soon after subculture, showing an additive stress effect of salt and light. In the PA − vs acclimated to high salinity, or immediately after stress application, the chloroplast content of chlorophyll a and b was considerably enhanced, like the TGase activity (by two-fold or more), and these changes exhibited almost coincident behaviours. Some transglutaminase substrates (proteins of 68, 55, 29 and 27 kDa) were found to be similar to those present in higher plants (thylakoid photosynthetic complexes and Rubisco). They were more markedly labelled by [1,4- 14 C] polyamines when the transglutaminase assay was performed in the light than in the dark, and much more in algae already acclimated to hyper-saline conditions than in those cultured in the optimal saline medium, or subjected to stress. The amount of 68 and 55 kDa polypeptides was particularly high in the 3.5 mol/L NaCl acclimated cells. The possible role of polyamine conjugation in the assembly of chloroplast proteins in cells affected by salt stress is discussed.

Jasmonate-induced ripening delay is associated with up-regulation of polyamine levels in peach fruit.

Methyl jasmonate (MJ, 0.20mM) and its synthetic analog n-propyl dihydrojasmonate (PDJ, 0.22mM) were applied to peach fruit (Prunus persica L. Batsch) at a late developmental stage under field conditions (in planta). On the basis of a previously demonstrated jasmonate (JA)-induced ripening delay in peach, the effects of JAs on the time course of the endogenous polyamine (PA) accumulation and expression of their biosynthetic genes arginine decarboxylase (ADC), ornithine decarboxylase (ODC), spermidine synthase (SPDS) and S-adenosylmethionine decarboxylase (SAMDC) were evaluated in control and JA-treated fruit during the 21-d trial period. In parallel, the main ripening-related parameters (ethylene production, flesh firmness and soluble solids contents) were measured, and transcription profiles of aminocyclopropane-1-carboxylic acid oxidase (PpACO1) and of two ethylene perception genes were evaluated. PDJ, but not MJ, reduced ethylene production and fruit softening, impaired PpACO1 transcription and altered the expression of PpERS1 (ethylene sensor 1), but not the expression of PpETR1 (ethylene receptor 1). In the epicarp and mesocarp, the pattern of PA accumulation was altered in a biphasic manner leading to a higher overall PA level in PDJ-treated fruit. Short and long term increases in putrescine, spermidine and/or spermine, the latter only in the epicarp, were observed in PDJ-treated fruit. MJ induced this behavior only with putrescine in the mesocarp. PpADC transcription was also enhanced soon after the PDJ treatment. Since PDJ-treated fruit were less ripe, their higher PA concentrations in treated fruit are discussed in light of the dual role of these molecules as stress/defense protective compounds and rejuvenating effectors.

Peach (Prunus persicaL.) fruit growth and ripening: transcript levels and activity of polyamine biosynthetic enzymes in the mesocarp

Transcript levels and activities of the polyamine biosynthetic enzymes arginine decarboxylase (ADC, EC 4.1.1.19), ornithine decarboxylase (ODC, EC 4.1.1.17) and S-adenosylmethionine decarboxylase (SAMDC, EC 4.1.1.21), as well as free polyamine titres, were analysed throughout the four growth stages S1-S4 leading up to ripening in the mesocarp from peach fruit (Prunus persica L. Batsch cv. Redhaven) grown under field conditions. SAMDC mRNA, which was northern analysed by using a PCR-generated homologous SAMDC probe, and ADC mRNA levels appeared quite stable during fruit development, while ODC transcript accumulation showed a discontinuous trend. The pattern of transcript levels during growth did not correlate with that of the relative enzyme activity, which instead correlated well with free polyamine levels. Both exhibited maximum levels in S1 and a smaller peak in S3. The behaviour of the polyamine biosynthetic machinery is discussed in relation to the different cell growth rates occurring during fruit development.

Tuber vegetative stages and cell cycle in Helianthus tuberosus : Protein pattern and their modification by spermidine

Summary This paper reports the protein amount and composition of tuber during its phases of formation, dormancy and sprouting. Parallely, the break of dormancy has been induced by excising slices of tuber parenchyma and treating them with 2,4-dichlorophenoxy acetic acid (2,4-D) to induce a new cell cycle; the protein composition, neosynthesis and post-translational modification by spermidine are reported during the phases of the cell cycle. From tuber formation to sprouting, protein content follows a bimodal trend. The protein bands were separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis and two main bands (38 and 55 kDa) were detected. Many bands, especially of high molecular mass, changed their amount during all phases of tuber vegetative period. With the progression of the cell cycle very high molecular mass proteins increased; neosynthesis and modification by polyamines could account for this increase. Parallelly, the amount of lower molecular mass bands decreased. Many proteins are differently synthesized in the various cell cycle phases. Modification by spermidine occurred post-translationally, evident by an 18 kDa band (which in fact does not incorporate methionine), whose possible identification is discussed. A role for newly synthesized transglutaminases in the protein modification by polyamines is evaluated in the light of the immunodetection of 58 and 90 kDa bands by polyclonal anti-transglutaminase antibody.

Auxin and ethylene interaction during fruit growth and ripening of Actinidia deliciosa

emission by inducing ACS and, recently, an interaction between ethylene and IAA on the expression of ACO genes was reported (Rasori et al., 2003). Moreover, recent data suggest that expression of endo-1,4-β-Dglucanase (EGase), expansin and polygalacturonase (PGase) isoforms are regulated by auxins during the cell expansion phase of growth, and by ethylene during ripening (Alexander and Grierson, 2002). In the present work, we investigated the effect of the 3,5,6-TPA on fruit growth and quality in Actinidia deliciosa (cv Hayward) and the interaction between the chemical and ethylene biosynthesis. In addition, expression studies of genes encoding protein involved in cell wall disassembly (PGase and expansin) were performed.

6-BA and NAA effect on ‘Galaxy’ fruit growth, abscission and quality: a comparison between the Po Valley and the South Tyrol producing areas

The present study concerned the fruit thinning efficacy of the cytokinin 6-benzyladenine (6-BA) at 150 ppm alone or in combination with the naphthalene acetic acid (NAA) at 15 ppm, on ‘Galaxy’ trees grown in two orchards, one in South Tyrol and the other in Emilia-Romagna area. Applications were carried out at different fruit sizes (5–7, 10–12, 14–16 mm of the king fruit diameter, KF Ø). In warm climatic conditions as in Emilia-Romagna, the best thinning efficacy was observed with the application of 6-BA alone when KF Ø reached 10–12 mm, while in South Tyrol, where climatic conditions are cooler than Emilia-Romagna, 6-BA alone showed its best thinning efficacy when KF Ø reached 14–16 mm. However, in this area, the best results were obtained with the combination 6-BA + NAA applied at 5–7 mm KF Ø. Moreover, all the chemical applications positively affected fruit quality traits at harvest and, in particular, soluble solid content and flesh firmness were higher in treated fruits as compared to control ones. In South Tyrol, the chemical thinners enhanced skin red colour.ZusammenfassungDie Fruchtausdünnungsfähigkeit des Cytokinins 6-Benzyladenin (6-BA) (150 ppm) allein oder in Kombination mit Naphthylessigsäure (NAA) (15 ppm) bei Apfelbäumen der Sorte ‘Galaxy’ wurde an zwei Standorten untersucht, in Südtirol und in der Region Emilia Romagna (Italien). Die Anwendungen wurden bei verschiedenen Fruchtgrößen durchgeführt (5–7, 10–12 oder 14–16 mm Apikalfruchtdurchmesser, KF Ø). Unter warmen klimatischen Bedingungen wie in der Emilia Romagna wurde der beste Ausdünnungseffekt durch einmalige Anwendung von 6-BA bei KF Ø 10–12 mm erzielt, während im kühleren Südtirol die stärkste Wirkung durch die Kombination von 6-BA mit NAA bei KF Ø 5–7 mm beobachtet wurde. 6-BA allein zeigte die beste Ausdünnung bei KF Ø 14–16 mm. Alle chemischen Ausdünnungsmaßnahmen hatten positive Auswirkungen auf die Fruchtqualität zum Erntezeitpunkt, wobei insbesondere der lösliche Feststoffgehalt und die Fruchtfleischfestigkeit erhöht waren. In Südtirol bewirkten die chemischen Ausdünner zudem eine Verbesserung der Deckfarbe der Früchte.

Nuclear DNA distribution and amitotic processes in activated Helianthus tuberosus tuber parenchyma

ABSTRACT The nuclear DNA content in dormant tubers of Helianthus tuberosus L. cv OB1 and in explants activated for several hours by 2,4-D was measured by in situ cytometry of Feulgen-stained nuclei and by flow cytofluorimetry of isolated DAPI-stained nuclei. The pattern of variation of mean nuclear DNA content was nearly the same using either method. During activation, DNA content reached a maximum at 9 h. Cytophotometric analysis of optical density profiles of the nuclei and molecular analysis of restricted DNAs indicated that the observed increase at 9 h is probably not related to chromatin decondensation due to a different pattern of cytosine methylation but rather to DNA synthesis. At 15 h amitoses occurred and generated pairs of similar sized nuclei, having an equal DNA content. Concomitantly, the class of nuclei with the highest DNA value disappeared whilst a class with a lower DNA content increased. Between 9 and 20–25 h, DNA content decreased, then increased up to 35 h, when a single DNA class acc...