In the present work it is shown that temporal changes in the endogenous PA complement, and in transcript abundance of ethylene biosynthesis and perception genes, as induced by exogenous Pu and Sd, in either the epicarp or mesocarp of nectarines, are associated with the slowing down of ripening, as confirmed by analogous effects exerted by AVG.
Pu and Sd concentrations are enhanced in treated fruit
In the fruit pericarp/mesocarp, endogenous concentrations of PAs followed the decreasing trend typical of a developing peach fruit (Ziosi et al., 2003), characterized by the four stages S1–S4 (Zanchin et al., 1994). PA concentrations were highest at the end of S1 (end of cell division phase), decreased sharply during S2 (slow growth by cell expansion and endocarp lignification), and gradually during S3 (rapid cell extension). At the end of S3, a minor peak in PA titres brought their values back to those of 20 d before. In the epicarp, PA titres were of the same order of magnitude and peaked in late S3 as in the mesocarp. Given the positive effect of indole-3-acetic acid (IAA) on polyamine synthesis (Torrigiani et al., 1987), this peak may be associated with a transient increase in IAA concentrations occurring in mid-S3 (Masia et al., 1992; Ziosi et al., 2003). Different from the mesocarp, the epicarp displayed a higher abundance of Pu relative to Sd; this may be regarded as a wound response (Perez-Amador et al., 2002) during sample preparation, particularly evident because the nectarine epicarp only consists of one cell layer (King et al., 1987).
Among PA treatments, Pu induced the most relevant and lasting increases in Pu titres, especially in the mesocarp. In the latter, the rise in Sd titres suggests that Pu was, to a certain extent, also transformed into the higher amine. On the other hand, the stimulatory effect of Sd on Pu concentrations in both epicarp and mesocarp might be a result of a feedback response to the triamine, which inhibits its own synthesis leading to nonutilized Pu accumulation (Hanfrey et al., 2002). In any case, endogenous Pu concentrations, although enhanced, were far lower than those exogenously applied. As discussed extensively elsewhere (Bregoli et al., 2002; Torrigiani et al., 2004), various factors may interfere with PA uptake and accumulation in fruit, such as mode of application, charge number and cell wall binding (Bagni & Torrigiani, 1992; Petkou et al., 2004; Bregoli et al., 2006); moreover, strong homeostatic mechanisms contribute to accommodate endogenous PA concentrations efficiently (Hanfrey et al., 2002; Mayer & Michael, 2003).
Aminoethoxyvinylglycine caused a substantial and lasting accumulation of Pu only in the epicarp, with minor effects on Sd concentrations, while, in agreement with previous work (Bregoli et al., 2002), it weakly and transiently counteracted the developmentally regulated Pu depletion in the mesocarp. Thus, AVG was more active on PA concentrations in the epicarp, while Pu and Sd exerted more influence on PA concentrations in the mesocarp. This may be because of a different cell mobility of AVG relative to PAs. Since the primary target of AVG is ACS activity (Huai et al., 2001), presumably the increase in Pu concentrations is the consequence of the inhibition of ethylene biosynthesis. In addition, AVG has been reported to inhibit diamine oxidase (DAO) activity, which is responsible for Pu catabolism (Torrigiani et al., 2003), and to induce some free Pu accumulation (Scaramagli et al., 1999) in tobacco. This AVG-induced increase in Pu titres may reinforce the inhibition of ethylene production and contribute to the ripening delay.
ADC and SAMDC messages are transiently reduced in treated fruit
The trend in PpADC mRNA abundance observed in the epicarp (more or less constant) and mesocarp (increasing) of control nectarines, before and at ripening, did not positively correlate with the physiological decrease in Pu concentrations. Equally, during peach fruit development, at least up to the onset of ripening, PpADC mRNA abundance did not correlate with either ADC activity or Pu concentrations (Ziosi et al., 2003). On the contrary, in developing apple, which differs from peach as regards climacteric ethylene emission, ADC message decreased until it became undetectable at ripening, while ODC was never detected (Hao et al., 2005a); this is in agreement with the fact that Pu concentrations gradually decrease during the same period (Biasi et al., 1991). Hao et al. (2005a) concluded that, in ripening apple, ADC was responsible for Pu synthesis. In the mesocarp of nectarines, only a weak PpODC message was detected. This is in agreement with previous results, which showed that ODC transcript abundance decreased during peach fruit development, while correlating positively with ODC activity and Pu accumulation (Ziosi et al., 2003). These results would suggest that ODC, instead of ADC, was responsible for Pu synthesis during development and up to ripening. However, based on present information, it is not possible to infer whether ADC or ODC is responsible for Pu synthesis in nectarine.
Multiple metabolic pathways interact to regulate Pu concentrations (Mayer & Michael, 2003), and it is important to note that agmatine is the product of arginine decarboxylation. Moreover, ADC transcript abundance does not always correlate directly with Pu concentrations, because of post-translational cleavage of the protein (Malmberg et al., 1992; Watson & Malmberg, 1996).
Further, ADC is prevalently involved in stress responses (Perez-Amador et al., 2002; Urano et al., 2003, 2004; Hummel et al., 2004) and ODC in active growth (Acosta et al., 2005). Indeed, ripening is considered a highly stressful physiological condition (Giovannoni, 2004). In Arabidopsis, perception and transduction of the ethylene signal may be involved in the transcriptional regulation of AtADC2, via the presence of numerous ERE (ethylene-responsive element) sequences in the promoter (Hummel et al., 2004). Since ADC promoters also respond to sucrose, in ripening nectarines, both ethylene and sucrose may trigger an increase in ADC message. The inhibitory effect of exogenous PAs on PpADC transcript abundance is in line with their effect on ripening delay, as shown by parallel effects on PpACO1, ethylene and quality patterns. Finally, in treated fruit, Pu and ADC transcript abundance was generally inversely correlated, suggesting that accumulation of the diamine triggers a feedback reduction of the latter.
In the epicarp of nectarines, PpSAMDC transcript was abundant and stable during fruit development and ripening. All treatments (especially AVG) negatively interfered with it, but while the Pu effect persisted, possibly because of its higher concentration, that of Sd and AVG was completely overcome at harvest, closely paralleling the effect of treatments on PpACO1. As in the case of PpADC, AVG was more active on PpSAMDC in the epicarp than in the mesocarp.
In the mesocarp, PpSAMDC message was transiently reduced but at harvest it increased again. The same was observed in ripening apple fruit and has been associated with the dramatic metabolic events occurring during the later stages of fruit ripening and abscission (Hao et al., 2005b). For instance, at this time, allergens are accumulated in many fruit species, including peach (Botton et al., 2002) with a probable role in defence against drought or pathogens. Given their role in stress responses, well documented in genetically engineered rice (Roy & Wu, 2001; Capell et al., 2004) and in Arabidopsis (Urano et al., 2003, 2004), both ADC and SAMDC could contribute to the establishment of the metabolic framework known as the ‘ripening syndrome’. Since both enzymes are post-transcriptionally and post-translationally regulated, the enhancement of their transcripts do not necessarily lead to PA accumulation, but can be ‘stored’ for future use.
Ethylene production and fruit softening are reduced in treated fruit
The higher Pu and Sd concentrations, especially in the mesocarp of PA-treated fruit, resulted in reduced ethylene production, retention of flesh firmness and reduction of fruit drop, confirming previous findings (in apricot, peach and nectarine) which have been discussed extensively elsewhere (Paksasorn et al., 1995; Bregoli et al., 2002; Torrigiani et al., 2004). It is worth noting that, in nectarines, earlier (4 wk before harvest) PA application than in the present work, at the same concentrations, markedly and persistently delayed ethylene production and fruit ripening (Torrigiani et al., 2004). The smaller reduction in ethylene production observed here relative to the latter work, might be attributed to the later PA application and fruit developmental stage. Indeed, PA treatment 7 d before harvest did not result in changes in ethylene production or fruit quality in nectarines (A. M. Bregoli, unpublished). It can be inferred that, in planta, the later the PA application, the weaker the effect on ripening. This may be the result of the fact that, when the molecular processes which underlie ripening are too far advanced, they cannot be effectively counteracted.
Treatments interfere with ethylene biosynthesis and perception
The dramatic increase in PpACO1 transcript abundance in both epicarp and mesocarp tissues during the time period considered is in accord with the progression of the climacteric peak. Although no PpACO2 signal was detected in the present work, in nectarines treated in postharvest with 1-methylcyclopropene (MCP), a specific inhibitor of ethylene perception, PpACO2, albeit much less expressed, followed the same trend as PpACO1 (Bregoli et al., 2005). In the epicarp, the rise in PpACO1 transcript abundance was counteracted, albeit with different timing, by PAs and AVG. The earlier and more lasting effect of Pu relative to Sd (and AVG) is in accord with its higher initial concentration and accumulation in treated fruit. In the mesocarp, the sharp increase in PpACO1 message accumulation was equally counteracted by treatments; again, at harvest, the message was partially (PAs) or totally (AVG) recovered. The latter lends support to the hypothesis, proposed in previous work, that the recovery of ACO transcript abundance to control values at harvest, in Sd-treated fruit, followed a previous inhibition (Torrigiani et al., 2004). This shift in ACO transcript accumulation may be interpreted as a slowing down of the transition from system 1, which is characterized by basal preclimacteric ethylene production and negative feedback regulation, to system 2, in which a high rate of ethylene production and a positive feedback regulation occur (Barry et al., 2000).
An important aspect of ethylene physiology is its perception and tissue sensitivity. Many genes have been isolated whose protein products bind ethylene, thus triggering the cascade of events of signal transduction. In this work, the expression of peach orthologs to ETR1 and ERS1 Arabidopsis genes was evaluated. In control nectarines, transcript accumulation of PpERS1 and PpETR1 showed opposite trends: the former increased while the latter decreased slightly during ripening. Only at harvest, in Pu-, Sd- and especially AVG-treated fruit, was an increase in PpERS1 (ethylene sensitive) transcript accumulation relative to controls observed. This response could be related to the rapid recovery in ethylene biosynthesis (see PpACO1 message), more accentuated in treated fruit than in controls, that is normally followed by an increase in receptor synthesis. In fact, when a plant actively synthesizes, or is exposed to a sudden increase in, a hormone, it responds by inducing mechanisms aimed at inactivating the response. Since, in the case of ethylene, receptors serve as negative regulators of the ethylene signal transduction pathway (Hua & Meyerowitz, 1998), an increase of ethylene receptor synthesis probably serves this purpose. A similar effect has been observed by Rasori et al. (2002) for Pp-ERS1 following treatment with 1-MCP, in agreement with results in ripening muskmelon and tomato fruit and in rice seedlings (Lashbrook et al., 1998; Sato-Nara et al., 1999; Yau et al., 2004).
The present results show that, besides Pp-ERS1, an increase in Pp-ETR1 also occurred in response to Sd and AVG. This could be the expression of a compensatory mechanism (existing within the complex of ethylene receptors) as observed in Never-ripe (NR)-antisense tomato plants where, besides the expected repression of NR transcript, Le-ETR4 was overexpressed (Tieman et al., 2000). Thus, the sudden recovery in ethylene biosynthesis presently observed at harvest in treated fruit seems to be actively counteracted by an increase in ethylene receptors.
In conclusion, exogenous Pu and Sd counteract, to various degrees, changes in climacteric ethylene production and fruit quality, resulting in a ripening delay/rejuvenating effect, probably by counteracting the physiological decrease in endogenous PA concentrations. In fact, despite strict homeostatic regulation, in PA-treated fruit PAs were maintained at concentrations compatible with much earlier developmental stages, in line with the antisenescence role of these molecules. Although AVG, whose inhibitory action on fruit ripening is well established, has a different metabolic target, its effects on ripening-related parameters (e.g. PpACO1 mRNA) are very similar to those of PAs, supporting the notion of the antithetical functional relationship between PAs and ethylene. Indeed, both PAs and AVG counteract climacteric ethylene production; however, inhibition is transient, and associated with an overproduction of specific receptors, probably in order to desensitize the tissue to the incoming recovery in ethylene production.