SEARCH

SEARCH BY CITATION

Keywords:

  • 1-aminocyclopropane-1-carboxylate oxidase;
  • aminoethoxyvinylglycine (AVG);
  • arginine decarboxylase;
  • ethylene receptors;
  • fruit ripening;
  • polyamines;
  • Prunus persica;
  • S-adenosylmethionine decarboxylase

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • • 
    The time course of ethylene biosynthesis and perception was investigated in ripening peach fruit (Prunus persica) following treatments with the polyamines putrescine (Pu) and spermidine (Sd), and with aminoethoxyvinylglycine (AVG).
  • • 
    Fruit treatments were performed in planta. Ethylene production was measured by gas chromatography, and polyamine content by high-performance liquid chromatography; expression analyses were performed by Northern blot or real-time polymerase chain reaction.
  • • 
    Differential increases in the endogenous polyamine pool in the epicarp and mesocarp were induced by treatments; in both cases, ethylene production, fruit softening and abscission were greatly inhibited. The rise in 1-aminocyclopropane-1-carboxylate oxidase (PpACO1) mRNA was counteracted and delayed in polyamine-treated fruit, whereas transcript abundance of ethylene receptors PpETR1 (ethylene receptor 1) and PpERS1 (ethylene sensor 1) was enhanced at harvest. Transcript abundance of arginine decarboxylase (ADC) and S-adenosylmethionine decarboxylase (SAMDC) was transiently reduced in both the epicarp and mesocarp. AVG, here taken as a positive control, exerted highly comparable effects to those of Pu and Sd.
  • • 
    Thus, in peach fruit, increasing the endogenous polyamine pool in the epicarp or in the mesocarp strongly interfered, both at a biochemical and at a biomolecular level, with the temporal evolution of the ripening syndrome.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Ripening control in peach is an important issue because, owing to early harvest and long storage times, quality of the fruit when it reaches the consumer is poor. Ripening is a complex and highly regulated phenomenon in which many endogenous and environmental factors are involved, all of which must be correctly balanced in order to achieve the best fruit quality (Giovannoni, 2004). Knowledge of the molecular mechanisms which underlie these interactions is fundamental in order to apply effective ameliorative strategies.

Ethylene plays a key role in climacteric fruit ripening, and manipulating its biosynthesis or perception, before or after harvest, may be useful for modulating the ripening process (Nakatsuka et al., 1998; Mathooko et al., 2001; Rasori et al., 2002; Bregoli et al., 2005). Ethylene synthesis starts from S-adenosylmethionine (SAM) and occurs through two steps: the first leads to 1-aminocyclopropane-1-carboxylic acid (ACC) via ACC synthase (ACS), and the second to ethylene via ACC oxidase (ACO), the latter including various members which are differentially expressed in different tissues and physiological stages. In peach, as in other climacteric fruit, both genes are highly expressed at ripening. Ethylene is perceived by specific receptors that in Arabidopsis are encoded by five genes, which are classified, based on differences in the deduced amino acid sequences, into two subfamilies. Subfamily I includes ETR1 (ethylene receptor 1) and ERS1 (ethylene sensor 1), while subfamily II consists of ETR2, ERS2 and EIN4 (Hua & Meyerowitz, 1998). In peach, ETR1 and ERS1 orthologs have been isolated and cloned. PpERS1 and PpETR1 show a similar organization to the corresponding genes in Arabidopsis, and a different expression pattern and ethylene sensitivity during peach fruit growth and ripening (Bassett et al., 2002; Rasori et al., 2002).

Among the substances capable of interfering with the ethylene biosynthetic machinery, there are the aliphatic polyamines (PAs) putrescine (Pu), spermidine (Sd) and spermine (Sm). These evolutionarily ancient polycations are growth regulators capable of interacting with and stabilizing nucleic acids and other negatively charged macromolecules (Cohen, 1998). In plants, they are absolutely required for regular cell division and differentiation (Bagni & Torrigiani, 1992) and for cellular homeostasis (Urano et al., 2003), as demonstrated with genetic engineering approaches (Hanfrey et al., 2002; Capell et al., 2004) or PA-defective mutants (Hanzawa et al., 2000; Urano et al., 2005). Putrescine derives from the amino acids arginine or ornithine via arginine (ADC) and ornithine decarboxylases (ODC), respectively, depending upon physiological conditions (Cohen, 1998); from this diamine, the higher PAs Sd and Sm are synthesized via a two-step pathway starting from SAM decarboxylase (SAMDC) through Sd and Sm synthases. The complex regulation of PA content also includes enzymes which conjugate and oxidize them. Fruit set, development and maturation are accompanied by dramatic changes in PA concentration and biosynthesis in relation to growth rate. In peach, apricot and apple, as well as in other fruit, PA concentration is highest after fertilization and during early growth, and lowest at ripening (Biasi et al., 1991; Paksasorn et al., 1995); such changes are paralleled by changes in transcript levels of their biosynthetic enzymes (Ziosi et al., 2003; Hao et al., 2005a,b).

Previous work has shown that exogenous PAs and aminoethoxyvinylglycine (AVG), the well-known inhibitor of ethylene biosynthesis (Yu & Yang, 1979; Huai et al., 2001), may interfere with fruit ripening. Field treatments with millimolar or submillimolar concentrations of Pu, Sd and Sm of peach and apricot fruit at a late developmental stage showed that these molecules interfere heavily with the time course of ethylene production, and with the development of quality parameters, without substantial alterations in endogenous PA concentrations at harvest (Paksasorn et al., 1995; Bregoli et al., 2002; Torrigiani et al., 2004). On the basis of these findings, and in order to clarify further how ripening-related genes, such as those encoding for ethylene biosynthesis and perception, are temporally modulated by PAs and AVG, the latter taken as a positive control, the aim of the present work was to monitor: (i) developmentally regulated changes in the endogenous PA pool of peach fruit epicarp and mesocarp; (ii) modifications induced by exogenous treatments on the endogenous PA pool and PA biosynthetic gene transcripts; and (iii) the time course of changes in transcript abundance of ACO, and of ETR1 and ERS1 in treated and untreated fruit during late development up to ripening. Results show that PAs and AVG, despite their different targets, induced highly comparable changes in metabolic parameters involved in the evolution of the ripening syndrome.

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Plant material and experimental design

The trial was carried out at the experimental farm of the Faculty of Agriculture of the University of Bologna, Italy. Experiments were performed on 6-year-old Stark Red Gold nectarine (Prunus persica L. Batsch, cv. Stark Red Gold) trees (20 per treatment), grafted on seedling rootstock, and trained to a Y-shape. Four branches per plant (total 80) were selected for experiments for uniform size and fruit load (three to four fruit per branch). For each treatment, 20 branches, including controls, were sprayed with ReTain® (ABG 3178, Valent Biosciences Corporation, Libertyville, IL, USA), a commercial product containing 15% (w/w) AVG a.i., or with 10 mm Pu or 0.1 mm Sd (Sigma-Aldrich, Milan, Italy), which were previously shown to be effective (Torrigiani et al., 2004). AVG (0.32 mm) was applied as an aqueous solution (150 ml per branch), containing 0.05% (v/v) nonionic surfactant (Silwet L-77®, ABG-7011, Valent Biosciences). PAs were dissolved in 50 mm Tris-HCl buffer, pH 7, and 150 ml per branch applied. All treatments were performed 15 d before harvest. The double sigmoid growth pattern of nectarines was established on the basis of fruit diameter, monitored twice a week on 40 fruit, and the first derivative was calculated in order to discriminate the four growth stages S1–S4 (Fig. 1; Torrigiani et al., 2004). For the purposes of this study, fruit were sampled before treatments (S1, S2 and S3), at the time of treatments (late S3), that is 123 d after full bloom (dAFB), and 3, 7, 15, 18 and 24 d after treatments. Ethylene production and fruit quality parameters were determined on whole fruits, while, for PA and molecular analyses, pericarp (S1–S2), or epicarp and mesocarp (S3–S4) tissues were stored separately at −80°C until use.

image

Figure 1. Peach (Prunus persica) fruit growth curve based on diameter and its first derivative from 30 to 138 d after full bloom (dAFB). S1–S4 are the four stages of growth up to harvest. Times of putrescine (Pu), spermidine (Sd) and aminoethoxyvinylglycine (AVG) treatments, and harvest are indicated by arrows.

Download figure to PowerPoint

Ethylene, fruit quality and abscission analyses

Ethylene evolution and the main quality traits were determined at 0, 3, 7 and 15 d after treatments in control and treated fruits. Ethylene production was measured by placing the whole detached fruit in a 1.7 l jar, sealed with an air-tight lid equipped with a rubber stopper, and left at room temperature for 1 h. A 10 ml gas sample was taken and injected into a Dani HT 86.01 (Dani, Milan, Italy) packed-gas chromatograph as previously described (Bregoli et al., 2002). Flesh firmness (FF) was measured using a pressure tester (EFFE.GI, Ravenna, Italy), and soluble solids concentration (SSC) was measured with an Atago digital refractometer (Optolab, Modena, Italy), as previously described by Bregoli et al. (2002). Fruit abscission was determined by counting all the dropped and still attached fruit at 15, 18 and 24 d following chemical applications in both control and treated plants.

HPLC polyamine determination

Pericarp, mesocarp or epicarp tissues from 10 randomly selected fruit were extracted in 4% perchloric acid (PCA) and centrifuged. The supernatant was dansylated and free PAs analysed by HPLC as previously described (Bregoli et al., 2002).

Isolation of peach ADC and ODC fragments

For the isolation of homologous ADC and ODC fragments, DNA was isolated from peach leaves following the protocol described in Doyle & Doyle (1990) with minor modifications, and 100 ng were amplified by using degenerate primers. For the PpADC1 probe, degenerated primers 5′-CT(A/G/C/T)NGARGCNGGNTCNAARCC-3′ (forward) and 3′-GGNCCNCCRAANAGRTTRTG-5′ (reverse) were designed based on the conserved amino acid regions of other plant ADCs. The PCR conditions were 94°C for 5 min, followed by 40 cycles of 95°C for 1 min, 54°C for 2 min and 72°C for 1.5 min. The PCR reaction produced multiple DNA fragments, the larger and more abundant of which was around the expected size (c. 1200 bp). This fragment was excised from the gel and subcloned into the pDRIVE vector (Qiagen, Milan, Italy) for sequencing. The sequenced product was 1268 bp in length and shared 100% similarity with the partial peach protein sequence present in the database (BAD 97829). The last portion of the amplified peach ADC (44 amino acids) was homologous to Malus × domestica (BAD 74163.1) and Vitis vinifera (CAA65585.1) ADCs available in the database.

For PpODC1, 50 ng of genomic DNA were PCR-amplified with the degenerated oligonucleotides 5′-CCNTTYTAYGCNGTNAARTGYAA-3′ (forward) and 5′-CCRTCRCANGTNGGNCCCCA-3′ (reverse). One-hundredth of the first-round product was used in a second round of PCR with the inner primers 5′-ATNTWYGCNAAYCCNTG-3′ (forward) and 5′-TNCCRTANACNCCRTCRTT-3′ (reverse). PCR conditions and thermal profiles were those described in Michael et al. (1996). The nested PCR produced a single DNA fragment of c. 600 bp. This fragment was subcloned into the pDRIVE vector (Qiagen) and sequenced. The sequenced product was 657 bp in length and was highly homologous to other plant ODCs present in the database (Prunus persica BAD97830.1; Populus nigra CAH60859.1; Vitis vinifera AAO49839.1).

RNA extraction and northern analysis

Total RNA was extracted from mesocarp or epicarp using the method described by Bonghi et al. (1998). RNA (18 µg per track) was size-fractionated and blotted onto nylon membranes (Hybond-N, Amersham Pharmacia Biotech Italia, Milan, Italy) according to standard methods (Sambrook et al., 1989) and hybridized with homologous probes.

The peach ACO1 (210 bp) and ACO2 (137 bp) probes were PCR-derived from the 3′ untranslated region as previously described (Bregoli et al., 2005). The peach SAMDC probe (1188 bp; accession number AJ704800) was obtained by PCR using primers designed on highly conserved regions of the SAMDC gene of Arabidopsis, as previously described (Ziosi et al., 2003). Following hybridization with ACO1, ACO2, ADC, ODC and SAMDC probes, membranes were washed and then exposed to the IP/Fluorescent Reader FLA-3000 Series (Fuji Photo Film, Tokyo, Japan) for 30 min (ACO1), 15 h (SAMDC), 5 h (ADC), 96 h (ACO2) and 15 d (ODC). Equal loading of gels was verified by ethidium bromide staining of RNA.

Quantitative RT-PCR

Three micrograms of total RNA were treated with 3 units of DNAse (Invitrogen Life Technologies, Carlsbad, CA, USA). The cDNA was obtained from 0.8 µg of DNAse-treated RNA using the Super Script III First Strand Synthesis System kit (Invitrogen Life Technologies, Strathclyde, UK) with the oligo-dT20 as a primer. Quantitative RT-PCR was performed using the SYBR® Green (RT)PCR Master Mix kit (Applied Biosystems, Foster City, CA, USA), as previously described by Rasori et al. (2002). Specific primers for Pp-ERS1 (sense 5′-GATTGAGAGTGAGGGCATTG-3′; antisense 5′-GCTGCTGTTGTATCACAAGG-3′), PpETR1 (sense 5′-ATGATAACGGGTCAGTGACT-3′; antisense 5′-AAATAACGTGCAAGAACTCATC-3′) and peach 18S rRNA (Nickrent & Soltis, 1995) (sense 5′-GTTACTTTTAGGACTCCGCC-3′; antisense 5′-TTCCTTTAAGTTTCAGCCTTG-3′) were designed to amplify fragments of 90 bp with a melting point of approx. 56°C. Three replicates were performed for each sample in 50 µl final volume containing 1 µl cDNA, 15 pmol PpETR1 or PpERS1 or 18S specific primers, and 25 µl of 2× SYBR Green PCR Master Mix according to the manufacturer's instructions. PCRs were carried out using the GeneAmp 5700 Sequence Detection System (AME Bioscience, Toroed, Norway) for 10 min at 95°C (initial denaturation) and then for 40 cycles as follows: 30 s at 95°C, 30 s at 54°C and 30 s at 72°C. For each sample, at the end of the PCR, the appropriate threshold was chosen, based on the fluorescence logarithmic graph, and the fractional cycle number at which a significant increase above the threshold could be first detected was calculated using the GeneAmp 5700 Sequence Detection System software. Data were obtained following the comparative method described in the User Bulletin No. 2 (Applied Biosystems). The amount of target was given as inline image, where ΔCt is the difference in threshold cycles for target (Ct sample) and reference (Ct 18S). The Ct for each sample was normalized to 18S rRNA.

Statistical analysis

Data on ethylene production and quality parameters represent the means (n = 40) and were analysed by anova procedures using the SAS Statistical Software (SAS Institute, Cary, NC, USA). Means were separated, between controls and treatments, and among treatments, using Duncan's multiple range test at the 5% level. Values expressed as a percentage (fruit drop) were analysed using the Kruskal–Wallis one-way anova to discriminate between groups followed by a pairwise multiple comparison (Dunn's or Tukey's method). Data on PA content were analysed by the one-way anova test (n = 9–12).

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Polyamine content during fruit development and after treatments

In the pericarp, PA titres decreased by one order of magnitude from the end of S1 through S2 and part of S3 (Fig. 2a). From 103 dAFB onwards, epicarp and mesocarp were analysed separately. In both tissues, the concentrations of the three amines continued to decrease gradually up to 118–120 dAFB, but then increased, giving rise to a minor peak around 124 dAFB (late S3; Fig. 2a,b). Thereafter, PA titres decreased again, so that at harvest they had returned to concentrations observed 20 d earlier. Their overall concentration was comparable in the two tissues, but their relative concentration differed: Pu predominated in the epicarp and Sd in the mesocarp.

image

Figure 2. Free polyamine titres in the pericarp (46–102 d after full bloom (dAFB)) or mesocarp (102–138 dAFB) (a) and epicarp (b) of peach (Prunus persica) fruit during development up to harvest. Pu, putrescine; Sd, spermidine; Sm spermine. S1–S4 indicate the four fruit growth stages. FW, fresh weight. 

Download figure to PowerPoint

Changes in PA concentrations as induced by exogenous Pu and Sd were monitored 3, 7 and 15 d following treatment. Minor changes were detected in the epicarp where, in controls, the gradual decrease in endogenous Pu concentrations was counteracted (significantly on day 7) by both Pu and Sd application (Fig. 3a). No significant changes in Sd (Fig. 3b) or Sm (data not shown) concentrations were detected relative to untreated fruit. On the contrary, AVG treatment led to a significant rise (more than twice control values) in Pu throughout (Fig. 3a), and to significant Sd accumulation compared with controls on day 7 (Fig. 3b). In the mesocarp, PA-induced differences were more marked and significant throughout the period following application, and were maximal on day 7 when Pu concentrations were up to fourfold higher in Pu-treated and twofold higher in Sd-treated fruit than in controls (Fig. 3c). Sd concentrations were maintained, on days 3 and 7, at approximately initial levels only in Pu-treated fruit (Fig. 3d), while Sm concentrations were never significantly affected by any treatment (data not shown). AVG only modestly enhanced Pu titres on day 7 (Fig. 3c).

image

Figure 3. Time course of free putrescine (Pu (a, c) and spermidine (Sd) (b, d) concentrations in the epicarp (a, b) and mesocarp (c, d) of peach (Prunus persica) fruit treated or not (control) with exogenous Pu (10 mm), Sd (0.1 mm) and aminoethoxyvinylglycine (AVG; 0.32 mm) during the 15 d following treatments. Data are means ± SD. Asterisks indicate significant differences compared with relative controls: *P < 0.05, **P < 0.01, ***P < 0.001. FW, fresh weight.

Download figure to PowerPoint

Time course of polyamine biosynthetic gene transcript abundance

In control fruit, ADC (2.8 kb) and SAMDC (1.9 kb) transcripts were detected, and their abundance varied in a differential manner in both epicarp and mesocarp. In the former, both mRNAs did not change substantially during the considered period (Fig. 4a,b). In treated fruit, in the epicarp, the ADC transcription pattern was only altered by AVG, which reduced transcript abundance as soon as day 3, and especially on day 7; at harvest, ADC mRNA recovered to above control values (Fig. 4a). In the same tissue, the SAMDC mRNA was increasingly reduced by Pu, while Sd inhibition, which was quite relevant, was confined to day 7 (Fig. 4b). As in the case of ADC, AVG markedly reduced the accumulation of SAMDC transcript on days 3 and 7 but, at harvest, the inhibition was removed, and the mRNA recovered to above control values.

image

Figure 4. Time course of transcript abundance of PpADC (a, d) and PpSAMDC (b, e) in the epicarp (a, b) and mesocarp (d, e) of untreated (control, C) peach (Prunus persica) fruit or fruit treated with putrescine (Pu, 10 mm), spermidine (Sd, 0.1 mm) or aminoethoxyvinylglycine (A, 0.32 mm) at 0, 3, 7 and 15 d after treatment. Northern blot analysis of total RNA (18 µg per lane) extracted from fruit epicarp and mesocarp, and relative ethidium bromide (EtBr)-stained loading controls.

Download figure to PowerPoint

In the mesocarp of control fruit, ADC mRNA abundance increased sharply on day 7 and then remained constant up to harvest. Transcript abundance was strongly and transiently reduced compared with controls by all treatments, with no differences between them, on day 7 (Fig. 4d). In the mesocarp, the pattern of SAMDC mRNA accumulation was quite different from that of the epicarp. In control fruit, transcript abundance decreased abruptly, reaching a minimum on day 7, but rose again at harvest (Fig. 4e). On day 3, PAs and AVG inhibited SAMDC transcript accumulation, while at harvest, in all treated fruit, transcript abundance was enhanced relative to controls.

While no ODC mRNA was detectable in the epicarp, a very weak ODC transcript (1.5 kb) was detected in the mesocarp; however, it did not allow detection of differences between controls and treated fruit, or among the treatments (data not shown).

Ethylene production, fruit quality and abscission

In control fruit, ethylene production was first detectable, although at very low quantities, 7 d following treatments, and increased by one order of magnitude up to harvest time (8 d later; Fig. 5a). Following treatments with PAs and AVG, ethylene production was significantly inhibited by Sd and AVG at 7 d and at harvest. All treatments were able to retain fruit firmness at harvest up to fourfold for Pu and approx. threefold for Sd and AVG relative to controls (Fig. 5b). By contrast, SSC did not change significantly in all treated compared with untreated fruit (11.6 ± 1.2°Brix on day 7 and 12.2 ± 0.7°Brix at harvest). Fruit abscission, evaluated 15, 18 and 24 d after treatments, was significantly reduced only on the latter day by both Sd and AVG (Fig. 5c).

image

Figure 5. Time course of ethylene production (a) and flesh firmness (b) in peach (Prunus persica) fruit, either untreated (control) or treated with putrescine (Pu, 10 mm), spermidine (Sd, 0.1 mm) or aminoethoxyvinylglycine (AVG, 0.32 mm) 15 d before harvest. (c) Percentage of fruit abscission 24 d after treatments. Data represent the means, and for every sampling time, different letters indicate significant differences at P < 0.05. FW, fresh weight.

Download figure to PowerPoint

Time course of transcript abundance of ethylene biosynthesis and perception genes

Temporal changes in PpACO1 gene expression were evaluated by northern analysis following PA and AVG application. In the epicarp of control fruit, PpACO1 mRNA abundance (1.4 kb) increased regularly throughout the 15 d period considered, and both PAs and AVG, albeit with different kinetics, counteracted this accumulation (Fig. 6a). The inhibitory effect of Pu was evident earlier, and was more lasting than that of Sd and AVG. In Sd- and AVG-treated epicarp, the increase in PpACO1 transcript accumulation was shifted by 8 d.

image

Figure 6. Time course of transcript abundance of PpACO1 in the epicarp (a) and mesocarp (b) of untreated peach (Prunus persica) fruit (control, C) or fruit treated with putrescine (Pu, 10 mm), spermidine (Sd, 0.1 mm) or aminoethoxyvinylglycine (AVG, 0.32 mm) at 0, 3, 7 and 15 d after treatment. Northern blot analysis of total RNA (18 µg per lane) extracted from fruit epicarp and mesocarp, and relative ethidium bromide (EtBr)-stained loading controls.

Download figure to PowerPoint

In the mesocarp of control fruit, PpACO1 mRNA abundance increased until harvest. The inhibitory effect of Pu and Sd was clearly detectable 7 d after application and persisted, albeit highly attenuated, up to harvest (Fig. 6b). AVG exerted a less marked effect than PAs on PpACO1 transcripts on day 7, while, at harvest, PpACO1 message recovered above control values. No PpACO2 signal was detectable in either the epicarp or the mesocarp.

A real-time RT-PCR evaluation was performed for the two ethylene receptors PpETR1 and PpERS1 in the mesocarp. PpERS1 mRNA abundance increased slightly in control fruit up to harvest (Fig. 7a); this pattern was not significantly altered by treatments on days 3 and 7 while, at harvest, ERS1 transcript was enhanced, especially in AVG-treated fruit (twofold). In control fruit, PpETR1 transcript slowly decreased up to harvest when it was stimulated by Sd and even more so by AVG (Fig. 7b).

image

Figure 7. Time course of the quantitative RT-PCR of PpERS1 (ethylene sensor 1) (a) and PpETR1 (ethylene receptor 1) (b) in the mesocarp of untreated peach (Prunus persica) fruit (control, C) or fruit treated with putrescine (Pu, 10 mm), spermidine (Sd, 0.1 mm) or aminoethoxyvinylglycine (A, 0.32 mm) at 0, 3, 7 and 15 d after treatment. The relative quantification of PpERS1 and PpETR1 transcript abundance was performed as described in the Materials and Methods section.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

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.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This research was supported by funds PRIN 2000 (20022078818-004) from the Italian MIUR to PT. The authors wish to thank Dr A. Rasori for criticism and helpful discussion, and Giulia Paganelli for her excellent contribution to the experimental work.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • Acosta C, Pérez-Amador MA, Carbonell J, Granell A. 2005. The two ways to produce putrescine in tomato are cell-specific during normal development. Plant Science 168: 10531057.
  • Bagni N, Torrigiani P. 1992. Polyamines: a new class of growth substances. In: KarssenCM Van Loon LC Vreugdenhil D, eds. Progress in plant growth regulation. Dordrecht, the Netherlands: Kluwer Academic Publishers, 264275.
  • Barry CS, Llop-Tous MI, Grierson D. 2000. The regulation of 1-aminocyclopropane-1-carboxylic acid synthase gene expression during the transition from system-1 to system-2 ethylene synthesis in tomato. Plant Physiology 123: 979986.
  • Bassett CL, Artlip TS, Callahan AM. 2002. Characterization of the peach homologue of the ethylene receptor, PpETR1, reveals some unusual features regarding transcript processing. Planta 215: 679688.
  • Biasi R, Costa G, Bagni N. 1991. Polyamine metabolism as related to fruit set and growth. Plant Physiology and Biochemistry 29: 497506.
  • Bonghi C, Ferrarese L, Ruperti B, Tonutti P, Ramina A. 1998. Endo-β-1,4-glucanases are involved in peach fruit growth and ripening, and regulated by ethylene. Physiologia Plantarum 102: 346352.
  • Botton A, Begheldo M, Rasori A, Bonghi C, Tonutti P. 2002. Differential expression of two lipid transfer protein genes in reproductive organs of peach (Prunus persica L. Batsch). Plant Science 163: 9931000.
  • Bregoli AM, Scaramagli S, Costa G, Sabatini E, Ziosi V, Biondi S, Torrigiani P. 2002. Peach (Prunus persica L.) fruit ripening: aminoethoxyvinylglycine (AVG) and exogenous polyamines affect ethylene emission and flesh firmness. Physiologia Plantarum 114: 472481.
  • Bregoli AM, Ziosi V, Biondi S, Bonghi C, Costa G, Torrigiani P. 2006. A comparison between intact fruit and fruit explants to study the effect of polyamines and aminoethoxyvinylglycine (AVG) on ripening in peach and nectarine (Prunus persica L. Batch). Postharvest Biology and Technology. (In press).
  • Bregoli AM, Ziosi V, Biondi S, Rasori A, Ciccioni M, Costa G, Torrigiani P. 2005. Postharvest 1-methylcyclopropene application in ripening control of ‘Stark Red Gold’ nectarines: temperature-dependent effects on ethylene production and biosynthetic gene expression, fruit quality, and polyamine levels. Postharvest Biology and Technology 37: 111121.
  • Capell T, Bassie L, Christou P. 2004. Modulation of the polyamine biosynthetic pathway in transgenic rice confers tolerance to drought stress. Proceedings of the National Academy of Sciences, USA 101: 99099914.
  • Cohen SS. 1998. A guide to the polyamines. New York: Oxford University Press.
  • Doyle A, Doyle G. 1990. Isolation of plant DNA from fresh tissues. Focus 12: 1315.
  • Giovannoni JJ. 2004. Genetic regulation of fruit development and ripening. Plant Cell 16: S170S180.
  • Hanfrey C, Franceschetti M, Mayer MJ, Illingworth C, Michael AJ. 2002. Abrogation of upstream open reading frame-mediated translational control of a plant S-adenosylmethionine decarboxylase results in polyamine disruption and growth perturbations. Journal of Biological Chemistry 277: 4413144139.
  • Hanzawa Y, Takahashi T, Michael AJ, Burtin D, Long D, Pineiro M, Coupland G, Komeda Y. 2000. ACAULIS5, an Arabidopsis gene required for stem elongation, encodes a spermine synthase. EMBO Journal 19: 42484256.
  • Hao Y-J, Kitashiba H, Honda C, Nada K, Moriguchi T. 2005a. Expression of arginine decarboxylase and ornithine decarboxylase genes in apple cells and stressed shoots. Journal of Experimental Botany 56: 11051115.
  • Hao Y-J, Zhang Z, Kitashiba H, Honda C, Ubi B, Kita M, Moriguchi T. 2005b. Molecular cloning and functional characterization of two apple S-adenosylmethionine decarboxylase genes and their different expression in fruit development, cell growth and stress responses. Gene 350: 4150.
  • Hua J, Meyerowitz EM. 1998. Ethylene responses are negatively regulated by a receptor gene family in Arabidopsis thaliana. Cell 94: 261271.
  • Huai Q, Xia Y, Chen Y, Callahan B, Li N, Ke H. 2001. Crystal structures of 1-aminocyclopropane-1-carboxylaste (ACC) synthase in complex with aminoethoxyvinylglycine and pyridoxal-5′-phosphate provide new insight into catalytic mechanisms. Journal of Biological Chemistry 276: 3821038216.
  • Hummel I, Bourdais G, Gouesbet G, Couée I, Malmberg RL, El Amrani A. 2004. Differential gene expression of ARGININE DECARBOXYLASE ADC1 and ADC2 in Arabidopsis thaliana: characterization of transcriptional regulation during seed germination and seedling development. New Phytologist 163: 519531.
  • King GA, Henderson KG, Lill RE. 1987. Growth and anatomical and ultrastructural studies of nectarine fruit wall development. Botanical Gazette 148: 433455.
  • Lashbrook CC, Tieman DM, Klee HJ. 1998. Differential regulation of the tomato ETR gene family throughout plant development. Plant Journal 15: 243252.
  • Malmberg RL, Smith KE, Bell E, Cellino ML. 1992. Arginine decarboxylase of oats is clipped from a precursor into 2-polypeptides found in the soluble enzyme. Plant Physiology 100: 146152.
  • Masia A, Zanchin A, Rascio N, Ramina A. 1992. Some biochemial and ultrastructural aspects of peach fruit development. Journal of the American Society of Horticultural Sciences 117: 808815.
  • Mathooko FM, Tsunashima Y, Owino WZO, Kubo Y, Inaba A. 2001. Regulation of genes encoding ethylene biosynthesis enzymes in peach (Prunus persica L.) fruit by carbon dioxide and 1-methylcyclopropene. Postharvest Biology and Technology 21: 265281.
  • Mayer JM, Michael AJ. 2003. Polyamine homeostasis in transgenic plants overexpressing ornithine decarboxylase includes ornithine limitation. Journal of Biochemistry 134: 765772.
  • Michael AJ, Furze JM, Rhodes MJC, Burtin D. 1996. Molecular cloning and functional identification of a plant ornithine decarboxylase cDNA. Biochemical Journal 314: 241248.
  • Nakatsuka A, Murachi S, Okunishi H, Shiomi S, Nakano R, Kubo Y, Inaba A. 1998. Differential expression and internal feedback regulation of 1-aminocyclopropane-1-carboxylate synthase, 1-aminocyclopropane-1-carboxylate oxidase, and ethylene receptor genes in tomato fruit during development and ripening. Plant Physiology 118: 12951305.
  • Nickrent DL, Soltis DE. 1995. A comparison of angiosperm phylogenies from nuclear 18S rDNA and rbcL sequences. Annals of the Missouri Botanical Garden 82: 208234.
  • Paksasorn A, Hayasaka T, Matsui H, Ohara H, Hirata N. 1995. Relationship of polyamine content to ACC content and ethylene evolution in Japanese apricot fruit. Journal of the Japanese Society for Horticultural Science 63: 761766.
  • Perez-Amador MA, Leon J, Green PJ, Carbonell J. 2002. Induction of the arginine decarboxylase ADC2 gene provides evidence for the involvement of polyamines in the wound response in Arabidopsis. Plant Physiology 130: 14541463.
  • Petkou JT, Pritsa TS, Sfakiotakis EM. 2004. Effects of polyamines on ethylene production, respiration and ripening of kiwifruit. Journal of Horticultural Science & Biotechnology 79: 977980.
  • Rasori A, Ruperti B, Bonghi C, Tonutti P, Ramina A. 2002. Characterization of two putative ethylene receptor genes expressed during peach fruit development and abscission. Journal of Experimental Botany 53: 23332339.
  • Roy M, Wu R. 2001. Arginine decarboxylase transgene expression and analysis of environmental stress tolerance in transgenic rice. Plant Science 160: 869875.
  • Sambrook J, Fritsch EF, Maniatis T. 1989. Molecular Cloning: a Laboratory Manual. Cold Spring Harbor: Cold Spring. Harbor Laboratory Press.
  • Sato-Nara K, Yuhashi KI, Higashi K, Hosoya K, Kubota M, Ezura H. 1999. Stage- and tissue-specific expression of ethylene receptor homolog genes during fruit development in muskmelon. Plant Physiology 120: 321330.
  • Scaramagli S, Biondi S, Capitani F, Gerola P, Altamura MM, Torrigiani P. 1999. Polyamine conjugate levels and ethylene biosynthesis: Inverse relationship with vegetative bud formation in tobacco thin layers. Physiologia Plantarum 105: 367376.
  • Tieman DM, Taylor MG, Ciardi JA, Klee HJ. 2000. The tomato ethylene receptors NR and LeETR4 are negative regulators of ethylene response and exhibit functional compensation within a multigene family. Proceedings of the National Academy of Sciences, USA 97: 56635668.
  • Torrigiani P, Bregoli AM, Ziosi V, Scaramagli S, Ciriaci T, Rasori A, Biondi S, Costa G. 2004. Pre-harvest polyamine and aminoethoxyvinylglycine (AVG) applications modulate fruit ripening in Stark Red Gold nectarines (Prunus persica L. Batsch). Postharvest Biology and Technology 33: 293308.
  • Torrigiani P, Scaramagli S, Castiglione S, Altamura MM, Biondi S. 2003. Downregulation of ethylene production and biosynthetic gene expression is associated to changes in putrescine metabolism in shoot-forming tobacco thin layers. Plant Science 164: 10871094.
  • Torrigiani P, Serafini-Fracassini D, Bagni N. 1987. Polyamine biosynthesis and effect of dicyclohexylamine during the cell cycle of Helianthus tuberosus tuber. Plant Physiology 84: 148152.
  • Urano K, Hobo T, Shinozaki K. 2005. Arabidopsis ADC genes involved in polyamine biosynthesis are essential for seed development. FEBS Letters 579: 15571564.
  • Urano K, Yoshiba Y, Nanjo T. 2004. Arabidopsis stress-inducible gene for arginine decarboxylase AtADC2 is required for accumulation of putrescine in salt tolerance. Biochemical and Biophysical Research Communications 313: 369375.
  • Urano K, Yoshiba Y, Nanjo T, Igarashi Y, Seki M, Sekiguchi F, Yamaguchi-Shinozaki K, Shinozaki K. 2003. Characterization of Arabidopsis genes involved in biosynthesis of polyamines in abiotic stress responses and developmental stages. Plant, Cell & Environment 26: 19171926.
  • Watson MW, Malmberg RL. 1996. Regulation of Arabidopsis thaliana (L.) Heynh arginine decarboxylase by potassium deficiency stress. Plant Physiology 111: 10771083.
  • Yu YB, Yang SF. 1979. Auxin-induced ethylene production and its inhibition by aminoethoxyvinylglycine and cobalt ion. Plant Physiology 64: 10741107.
  • Yau CP, Wang L, Yu M, Zee SY, Yip WK. 2004. Differential expression of three genes encoding an ethylene receptor in rice during development, and in response to indole-3-acetic acid and silver ions. Journal of Experimental Botany 55: 547556.
  • Zanchin A, Bonghi C, Casadoro G, Ramina A, Rascio N. 1994. Cell enlargement and cell separation during peach fruit development. International Journal of Plant Sciences 155: 4956.
  • Ziosi V, Scaramagli S, Bregoli AM, Biondi S, Torrigiani P. 2003. Peach (Prunus persica L.) fruit growth and ripening: transcript levels and activity of polyamine biosynthetic enzymes in the mesocarp. Journal of Plant Physiology 160: 11091115.