Physiologia Plantarum 128: 351–359. 2006
Copyright ª Physiologia Plantarum 2006, ISSN 0031-9317
Interrelationship between polyamine and ethylene in 1-methylcyclopropene treated apple fruits after harvest Xiao-Ming Panga, Kazuyoshi Nadab, Ji-Hong Liua,1, Hiroyasu Kitashibaa, Chikako Hondaa, Hiroyuki Yamashitac, Miho Tatsukia, Takaya Moriguchia,* a
Department of Plant, Cell and Environment, National Institute of Fruit Tree Science, Tsukuba, Ibaraki 305-8605, Japan Faculty of Bioresources, Mie University, Tsu, Mie 514-8507, Japan c Nagano Chushin Agricultural Experimental Station, Shiojiri, Nagano 399-6461, Japan b
Correspondence *Corresponding author, e-mail:
[email protected] Received 12 December 2005; revised 30 March 2006 doi: 10.1111/j.1399-3054.2006.00748.x
To investigate the interrelationship between polyamine and ethylene, apple fruits (Malus domestica Borkh.) were treated with 1-methylcyclopropene (1-MCP). Without 1-MCP treatment, ethylene production of apple fruits rapidly increased to maximum level at 4 days after treatment (DAT) and kept its level until 21 DAT, then gradually decreased. Accordingly, fruit firmness was progressively reduced until 26 DAT. On the other hand, ethylene production in 1-MCP-treated fruits was almost completely repressed until 21 DAT, retaining fruit firmness. This ethylene inhibition by 1-MCP was accompanied by the reduction of transcript accumulations for 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS) and ACC oxidase (ACO). The expression of MdSAMDC1 showed no differences between 1-MCP-treated and control fruits, whereas that of MdSAMDC2 was rather repressed in 1-MCP-treated fruits compared with the control. There were no statistically significant differences of free spermidine (Spd) and spermine (Spm) titers between fruits with and without 1-MCP treatment; however, Spd titer in 1-MCP-treated fruits temporally increased in the early DAT, then was adjusted to similar level as that in control, whereas Spm level was always higher in 1-MCP-treated fruits than in the control, indicating that Spd may be under a tight homeostatic regulation, whereas Spm may not. Therefore, the results demonstrated that the antagonistic relationships between polyamine and ethylene might exist transiently in the early days after 1-MCP treatment, but disappear later possibly because of the polyamine homeostasis mechanism and the extension of apple shelf life by 1-MCP might be owed little to the role of polyamines and be mainly ascribed to the inhibitory effects of ethylene on modulation of all wall.
Abbreviations – 1-MCP, 1-methylcyclopropene; ACC, 1-aminocyclopropane-1-carboxylic acid; ACO, 1-aminocyclopropane1-carboxylic acid oxidase; ACS, 1-aminocyclopropane-1-carboxylic acid synthase; cDNA, complementary DNA; DAT, days after treatment; DIG, digoxigenin; FW, fresh weight; Put, putrescine; RT-PCR, reverse transcriptase–PCR; SAM, S-adenosylmethionine; SAMDC, S-adenosylmethionine decarboxylase; SE, standard error; Spd, spermidine; Spm, spermine. 1
Present address: National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, China
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Introduction For climacteric fruits, ethylene is a key regulatory molecule for ripening and senescence and it is thought to regulate fruit ripening by coordinating the expression of many genes, which are responsible for chlorophyll degradation, carotenoid synthesis, conversion of starch to sugars, cell-wall modulating and so on (Theologis 1992). Polyamines, commonly among which are putrescine (Put), spermidine (Spd) and spermine (Spm), are small positively charged amines that are ubiquitous in living cells and are involved in many cellular processes such as DNA replication, transcription, translation and cell proliferation (Tabor and Tabor 1984). In plants, polyamines have been reported to play a crucial role in morphogenesis, embryogenesis, early fruit development and elicitation of resistance or tolerance responses to some biotic and abiotic stresses (Bouchereau et al. 1999, Galston and Sawhney 1990, Pandey et al. 2000). Polyamines have also been implicated as having roles in fruit ripening and senescence, which may be partly because of shifting demands for S-adenosylmethionine (SAM) between ethylene and polyamine biosynthesis because SAM is a common precursor for both biosynthetic pathways. Biosynthesis of ethylene is proceeded by conversion of SAM to 1-aminocyclopropane-1-carboxylic acid (ACC) by ACC synthase (ACS) and then to ethylene by ACC oxidase (ACO) (Oetiker and Yang 1995). SAM is also catalyzed by S-adenosylmethionine decarboxylase (SAMDC) into decarboxylated SAM, which provides an aminopropyl moiety for Spd and Spm biosynthesis. There have been many reports examining the relationship between ethylene and polyamines so far. Indeed, a correlation between polyamine-mediated modification of ethylene metabolism and antisenescence effects has been demonstrated in different plant tissues (Even-Chen et al. 1982, Ke and Romani 1988, Li et al. 1992, Roberts et al. 1984), although controversial reports on competing effects of polyamine and ethylene are also available (Escribano and Merodio 1994, Kushad et al. 1988). Thus, it seems that the interrelationship between polyamine and ethylene may vary with species, type of tissue and the experimental system used (Wang et al. 1993). In apple, it has been reported that polyamine- and ethylene-biosynthetic pathways are not actively competing based on the exogenous application of polyamines to apple fruits (Kramer et al. 1991, Wang et al. 1993). However, there have been no reports on the polyamine and ethylene changes in apple fruits when ethylenebiosynthetic pathway was blocked. For inhibiting ethylenebiosynthetic pathway, aminoethoxyvinylglycine and aminooxyacetic acid have been generally used as seen in carnation flower (Roberts et al. 1984), Japanese pear 352
(Ohkawa et al. 1998), peach (Bregoli et al. 2002), kiwifruit (Arigita et al. 2004) and nectarines (Torrigiani et al. 2004). Recently, 1-methylcyclopropene (1-MCP), an inhibitor of ethylene action was developed. 1-MCP is superior to other inhibitors in its low toxic and high-binding features to ethylene receptor, and, therefore, it was found promising to increase postharvest life and maintain fruit quality (Blankenship and Dole 2003, Serek et al. 1994). Actually, prolonging of storage period with 1-MCP has been reported in many horticultural products including apple (Fan et al. 1999, Mir et al. 2001, Pre-Aymard et al. 2003, Watkins et al. 2000). This prolonging storage period has been ascribed possibly to the inhibition of ethylene production through feedback inhibition as demonstrated in apple and tomato (Hoeberichts et al. 2002, Nakatsuka et al. 1997). On the other hand, 1-MCP can also induce ethylene production in some crops (cited in Blankenship and Dole 2003). In this study, we intended to evaluate the effects when ethylene pathway was blocked by 1-MCP for better understanding of the interrelationship between polyamine and ethylene in apple fruit ripening and senescence. We monitored ethylene production, loss of fruit firmness, weight loss, polyamine titer, ACC contents and expression of the ethylene- and polyamine-biosynthetic genes. The results show that 1-MCP can retain apple fruit firmness and inhibit ethylene production by repressing the expression of the ethylenebiosynthetic genes and that the antagonistic relationships between polyamine and ethylene might exist transiently in the early stage after 1-MCP treatment, which, however, disappear in the later stage possibly because of the polyamine homeostasis mechanism.
Materials and methods Plant materials and treatments Apple fruits (Malus domestica Borkh. ‘Orin’) were purchased from commercial orchard in Nagano prefecture, Japan. Fruits without any injured symptoms were selected and temporally stored at 4C for 16 h and transferred to 24C. For 1-MCP treatment, the fruits were treated with 1.0 ml l21 of SmartFreshTM (AgroFresh, Inc. Spring House, PA) at 24C for 16 h in a sealed plastic container. After 1-MCP treatment, fruits were then transferred to 24C. Control fruits without exposure to 1-MCP were also transferred from 4 to 24C conditions. Five fruits from each treatment of the samples returned to 24C were sampled at the indicated times. These fruits were first used for ethylene production and then for flesh firmness. The mesocarp of the same fruits was pooled and immediately frozen in liquid nitrogen and stored at 280C for subsequent use for ACC and polyamines measurement and RNA isolations. Physiol. Plant. 128, 2006
Determination of fruit firmness, weight loss, ethylene, ACC and polyamines Fruit firmness was measured by a penetrometer (Effegi, Italy) with an 8-mm diameter probe after removing a small disk of skin from the opposite sides of the fruit. Weight loss was calculated as percentage of fresh weight (FW) loss against the initial weight (before treatment) at each sampling time. Ethylene was measured by placing each fruit in a sealed desiccator (ca. 1500 ml) for 1 h at 25C. A 1.0 ml sample of the gas was withdrawn with a syringe and injected into a gas chromatograph (GC-14B Shimadzu, Japan) equipped with a flame ionization detector. Helium was used as a carrier gas, and activated alumina as column packing. The column and injector temperatures were set at 80C. ACC was extracted and quantified by the method of Lizada and Yang (1979) with 80% ethanol extracts from frozen tissues, which were partially purified on cationexchange resin (AG 50W-X8, Bio-Rad, Hercules, CA) column. ACC contents were expressed in nmol g21 as mean standard error (SE) of three replicates. Polyamines in apple were extracted and measured as described by He et al. (2002). Polyamine contents were expressed in nmol g21 as mean SE of three replicates.
using the BigDyeTM Terminator Cycle Sequencing Ready Reaction kit (PerkinElmer Life Sciences, Japan), these clones were designated as Md-ACS1 (Malus domesticaACS1) and pMdACO (partial Malus domestica ACO), respectively. Probes specific to apple SAMDC, MdSAMDC1 and MdSAMDC2, respective cell growthand stress-related SAMDCs, were previously produced by PCR (Hao et al. 2005). Subsequently, four kinds of cDNAs, Md-ACS1, pMDACO, MdSAMDC1 and MdSAMDC2, were labeled with digoxigenin (DIG)-dUTP by PCR (Roche Diagnostics, Mannheim, Germany) and used as probes. RNA gel blot analysis Total RNA was isolated from about 10 g of pooled fruit flesh tissues. Five micrograms of RNA was electrophoresed in 1.2% formaldehyde denatured agarose gel and blotted onto Hybond N membrane (Amersham Biosciences) and hybridized with DIG-labeled probes as described by Zhang et al. (2003). Signal intensities of hybridization were normalized with Scion Image (Scion Corporation, Frederick, MD) against the hybridization signal. Statistical analysis
Probe preparation for RNA gel blot analysis Total RNA was isolated from apple shoots (‘Orin’) according to the procedure described by Wan and Wilkins (1994), and 4 mg of total RNA was used to synthesize first-strand complementary DNA (cDNA) with a cDNA synthesis kit (Amersham Biosciences, Piscataway, NJ). A full-length ACS cDNA fragment was obtained by reverse transcriptase (RT)–PCR (StrataScriptTM RT-PCR kit, Stratagene, La Jolla, CA) using an amplification profile containing one cycle of 10 min at 94C, 30 cycles of 30 s at 94C, 30 s at 45C and 90 s at 72C and one cycle of 10 min at 72C. Two sets of primers 5#-CTT ACA GC T TGT ATC CAT ACA CAA G-3# (upstream) and 5#-TAC ACT AAT CAC ATT GTA TAG AAT C-3# (downstream) were designed based on the conserved regions of ripening-related apple ACS (Sunako et al. 1999). Isolation of ACO was also carried out according to the same method as described for ACS using total RNA from apple fruit (Wan and Wilkins 1994). A partial ACO cDNA fragment was amplified using two specific primers 5#-TCC CAG TTG TTG ACT TGA GCC-3# (upstream) and 5#-TGA GTC GTT GCC TGG GTT GT-3# (downstream) based on the ripening-related apple ACO sequences (Ross et al. 1992). Both amplified fragments were ligated into pCR2.1 vector (Invitrogen, Carlsbad, CA). After the homologies were confirmed by sequencing Physiol. Plant. 128, 2006
The data obtained were subjected to one-way analysis of variance test using ‘‘Data analysis’’ tool in Excel (Microsoft, Japan).
Results Fruit firmness and weight loss Fruit firmness of the control fruits decreased sharply from 4 days after treatment (DAT) to 17 DAT, after which it gradually decreased to 1.9 kg (Fig. 1A). In the 1-MCPtreated fruits, loss of firmness was significantly inhibited and the fruit firmness was maintained at about 4.0 kg. When held at 24C, weight loss in the control fruits was greater than that of the 1-MCP-treated fruits; however, the differences were not statistically significant and it seems that weight loss in 1-MCP-treated fruits showed similar trend as that of the control fruits (Fig. 1B). Ethylene production and ACC content Ethylene production in control fruits rose notably to its maximum level at 4 DAT and retained its level until 21 DAT, then decreased (Fig. 2A). Contrary to the control, ethylene production in the 1-MCP-treated fruits was barely detected until 21 DAT (Fig. 2A), when it began to 353
Fig. 1. Fruit firmness (A) and weight loss (B) in apple held at 24C after treatment with or without 1-methylcycropropene. Bars indicate standard error (n ¼ 5). Asterisks indicate significance level at **P < 0.01. Cont, control.
increase, indicating the efficiency of 1-MCP for ethylene inhibition in apple fruits. ACC content in the control varied with a value between ca. 1 and 16 nmol g21 FW, whereas it was not detected in the 1-MCP-treated fruits with the exception of ca. 1.2 nmol g21 FW at 35 DAT (Fig. 2B), paralleling to the ethylene production pattern. ACS and ACO transcript levels To investigate the inhibitory effects of ethylene biosynthesis by 1-MCP, RNA gel blot analyses of ACS and ACO were carried out. Because various isoforms for ACS and ACO genes were present, we isolated ripeningrelated types by RT-PCR based on previous reports (Ross et al. 1992, Sunako et al. 1999). In the control, Md-ACS1 expression increased from 4 to 21 DAT, and then declined slightly (Fig. 3), which concurred with the decline in ethylene (Fig. 2A). On the other hand, the transcription of Md-ACS1 in 1-MCP-treated fruits has been inhibited completely before 31 DAT and it reaccumulated at 35 354
Fig. 2. Ethylene production (A) and 1-aminocyclopropane-1-carboxylic acid (ACC) content (B) in apple held at 24C after treatment with or without 1-methylcyclopropene. Bars indicate standard error (se) (n ¼ 5) and se (n ¼ 3) for ethylene production and ACC content, respectively. Asterisks indicate significance level at *P < 0.05 and **P < 0.01. Cont, control.
DAT (Fig. 3), with the increase in ACC content (Fig. 2B) and ethylene production (Fig. 2A). The transcript level of pMdACO in control increased at 4 DAT and remained elevated level until 31 DAT (Fig. 3), followed by decrease to original level at harvest. In the 1-MCP-treated fruits, pMdACO transcripts were markedly lower than in the control before 31 DAT, when they showed no change or were repressed (Fig. 3), and they began to increase from 31 DAT and accumulated to higher level at 35 DAT. Changes in free polyamine titers As shown in Fig. 4, all three common polyamines, Put, Spd and Spm, were detected and Spd was the main free polyamines. Put markedly declined in both 1-MCPtreated and control fruits without significant difference except at 4 DAT, when it was higher in 1-MCP-treated fruit than in control fruits (Fig. 4A). Spd titer increased at 4 Physiol. Plant. 128, 2006
Discussion
Fig. 3. Expression of Md-ACS1and pMdACOgenes in apple held at 24C after treatment with or without 1-methylcyclopropene. The relative value of the expression level that was corrected to the ribosomal RNA level by densitometric analysis was indicated in lower panel. The hybridization signal in the sample before treatment was set to 100% and the others were quantified.
DAT in 1-MCP-treated fruits and was higher than those in the control before 17 DAT and lower thereafter (Fig. 4B), although the differences were not statistically significant. Spm titer in 1-MCP-treated fruits was higher than that in the control during the whole period (Fig. 4C), albeit not statistically significant. The changes of total polyamines levels in the control and 1-MCP-treated fruits (Fig. 4D) were similar to the pattern of change in Spd (Fig. 4B). SAMDCs transcript levels In our previous report (Hao et al. 2005), we successfully isolated two types of SAMDC genes, MdSAMDC1 and MdSAMDC2, which were identified as cell growth– and stress-related genes, respectively. The MdSAMDC1 messenger RNA levels showed no obvious differences between the 1-MCP-treated fruits and the control, whereas the accumulation of MdSAMDC2 transcript was repressed in the 1-MCP-treated fruits compared with the control (Fig. 5). Physiol. Plant. 128, 2006
It has been deemed that two systems of ethylene production exist in higher plants (McMurchie et al. 1972, Oetiker and Yang 1995). System 1 is the basal low rate of ethylene production present in non-climacteric fruits, in the preclimacteric stage of climacteric fruits and in vegetative tissues. System 2 presents the high rate of autocatalytic ethylene production observed during climacteric stage in the ripening of climacteric fruits and in certain senescent flowers. 1-MCP treatment caused complete repression of ethylene production in apple fruits within the early days when they were held at 24C. The suppression of ethylene was possibly resulted from feedback inhibition, which was in accord with the repression of Md-ACS1 expression and ACC accumulation by 1-MCP. The almost complete suppression of ACS accumulation with 1-MCP treatment has also been reported in tomato (Nakatsuka et al. 1997) and banana (Pathak et al. 2003). The level of pMdACO transcript was moderately reduced in 1-MCP-treated fruits; however, it was expressed constitutively and the level of pMdACO transcript increased when the fruit emitted ethylene again, similar to observations in banana (Pathak et al. 2003). It appears that the concept of two systems of ethylene production could be explained by the differential expression of individual members of gene families ACS and ACO (Katz et al. 2004, Nakatsuka et al. 1998, Oetiker and Yang 1995). Unfortunately, these two gene families have not been analyzed in detail during apple fruit ripening. We herein investigated only one member for each gene family, which was confirmed to be ripening related. It warrants further efforts to reveal the full scenario of ACS and ACO gene families in apple fruit ripening. 1-MCP-treated fruits started to emit ethylene again at 21 DAT, which induced obvious reaccumulations of MdACS1 and pMdACO transcripts at 35 DAT, indicating that multiple application of 1-MCP may be needed to achieve desirable duration of shelf life. The recovery of ethylene sensitivity may be because of either the release of previously occupied ethylene receptors or the synthesis of new receptors. Blankenship and Dole (2003) and Nakatsuka et al. (1997) suggested the possibility for the new synthesis of receptors, although the direct evidences are lacking. Fruit firmness is an important indicator for quality of postharvested apples. The firmer fruits are considered to have better quality than the softer fruits (Harker et al. 1997). In this study, 1-MCP could retain fruit firmness of ‘Orin’ held at 24C as reported in other apple cultivars (Fan et al. 1999, Mir et al. 2001, Pre-Aymard et al. 2003, Watkins et al. 2000). It has been suggested that ethylene acts as a rheostat rather than a trigger for fruit ripening, 355
Fig. 4. Free putrescine (A), spermidine (B) and spermine (C) titers and total free polyamines (D) in apple held at 24C after treatment with or without 1-methylcyclopropene. Bars indicate standard error (n ¼ 6). Asterisks indicates significance level at *P < 0.05. Cont, control.
indicating that ethylene must be present continuously to maintain transcription of the necessary genes (Davies et al. 1988, Theologis 1992). Hoeberichts et al. (2002) further confirmed that ethylene perception is required for the expression of tomato ripening-related genes even at advanced stages of ripening. Although herein cell-wall modulating genes were not monitored, suppression of expansin 1 (Hoeberichts et al. 2002) and polygalacturonase (Jeong et al. 2002) by 1-MCP treatment was reported. Thus, the effect of 1-MCP on the retention of apple fruit firmness should be at least in part ascribed to the inhibition of ethylene production. It is clear that many other factors other than ethylene have a controlling effect on fruit-ripening process. For instance, it is thought that polyamines can also contribute to fruit ripening, possibly because of their ability to stabilize and protect membranes (Slocum et al. 1984). Mehta et al. (2002) demonstrated that polyamines indeed had a function in delaying the ripening process using transgenic tomato. In apple, exogenous application of polyamines improved fruit firmness without affecting ethylene pathway (Kramer et al. 1991, Wang et al. 1993). Therefore, Kramer et al. (1991) proposed that polyamines affected fruit softening through rigidification of cell walls rather than through interaction with ethylene synthesis. Polyamine treatment delayed flesh softening in nectarine; however, no changes 356
in the endogenous polyamine pool were detected, implying that the control of flesh firmness of nectarine exerted by polyamines was mediated by ethylene (Torrigiani et al. 2004). In this study, inhibition of ethylene pathway also retained apple fruit firmness without statistically significant changes of polyamine titers, implying little contribution of polyamine to the extension of apple shelf life by 1-MCP. One might suppose that the weight loss was also prevented because of the delayed loss of firmness. Unexpectedly, the weight loss, however, could not be alleviated greatly in the 1-MCP-treated apple fruits. Furthermore, the 1-MCP-treated fruits seemed more spongy than the control from 14 DAT, possibly because of the subtle changes in cell-wall compositions through influencing water balance in apple fruits. Because SAM is a common precursor for the biosynthesis of polyamine and ethylene, a competition between polyamine and ethylene biosynthesis for utilization of SAM has been postulated (Apelbaum et al. 1981, Even-Chen et al. 1982). The supporting evidence was mainly from the opposite accumulating patterns of these two components as shown in Alcobaca tomato fruit (Dibble et al. 1988). Roberts et al. (1984) also demonstrated the reverse accumulation using inhibitors of both polyamine and ethylene biosyntheses in carnation Physiol. Plant. 128, 2006
Fig. 5. Expression of MdSAMDC1and MdSAMDC2genes in apple held at 24C after treatment with or without 1-methylcyclopropene. The relative value of the expression level that was corrected to the ribosomal RNA level by densitometric analysis was indicated in lower panel. The hybridization signal in the sample before treatment was set to 100% and the others were quantified.
flowers. Furthermore, the antisense SAMDC transgenic plant had greatly increased level of ethylene, also providing the support for the competitive relationships (Kumar et al. 1996). On the other hand, poor correlations between them were also available as revealed in some tissues, in which treatment with polyamines resulted in the stimulation of ethylene (Pennazio and Roggero 1990). The concurrent increases in polyamines and ethylene were found during mango fruit ripening, which may suggest that their biosynthesis may not be competitive, and polyamines may evolve as a response to increased ethylene production (Malik and Singh 2004). Transgenic poplar cell lines (Quan et al. 2002) and transgenic tomato (Mehta et al. 2002) also suggested that the SAM pool was not limiting for either pathway. Thus, the relationship between polyamine and ethylene seem to be largely dependent on the plant species. If competitive relationship was present between ethylene and polyamine biosynthesis, it would be expected that polyamine titers, i.e. Spd and/or Spm, should increase in the 1-MCP-
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treated fruits. Indeed, Spd and Spm titers were initially higher in 1-MCP-treated fruits held at 24C compared with those of the control. This increase in Spd and Spm takes place likely through the activation of polyaminebiosynthetic pathway, possibly inductions of SAMDC transcript and SAMDC activity when internal ethylene was inhibited. However, SAMDC transcripts, especially MdSAMDC2, were rather reduced in 1-MCP-treated fruits. Similar results have been reported in nectarines held at 24C after 1-MCP treatment (Bregoli et al. 2005). MdSAMDC1 was less affected than MdSAMDC2 after 1MCP treatment, which is in agreement with the inference that MdSAMDC1and MdSAMDC2 may differentially function in developmental and physiological process (Hao et al. 2005). Herein, the repression of the MdSAMDC2 expression did not coincide with the changes of polyamine titer, which may be because of posttranscriptional or translation regulation on SAMDC in apple fruits as seen in mustard (Hu et al. 2005) and in Arabidopsis (Hanfrey et al. 2002). One possible reason for higher levels of Spd and Spm in 1-MCP-treated fruits was that more SAM was channeled to Spd/Spm biosynthesis when ethylene biosynthesis was inhibited. It has been recently proposed that Spd and Spm in the cells are under a tight homeostatic regulation (Bhatnagar et al. 2002). In our studies, it is likely that the fruit cells adjusted their metabolism that restored Spd to normal levels. But this did not happen with Spm levels. That Spm levels remain consistently higher in 1-MCP-treated fruits is an interesting observation. The reason for this needs to be ascertained in future experimentation. Therefore, the present study demonstrated that the antagonistic relationships between polyamine and ethylene exist transiently soon after 1-MCP treatment, which is not, however, sustained with regard to Spd. To better understand the relationship between polyamine- and ethylene-biosynthetic pathways in apple fruits ripening, an investigation using methylglyoxal(bis-guanylhydrazone), an inhibitor of SAMDC is under way to repress polyamine pathway. Furthermore, monitoring of SAM biosynthesis pathway and recycling may also help decipher this relationship because it has been reported that the plant has a high plasticity in regulatory mechanisms of SAM pool (Ravanel et al. 1998). The use of molecular genetics approach may be a preferred way to directly answer this question.
Acknowledgements – Authors thank Mr. Yoshiki Kashimura (National Institute of Fruit Tree Science) for his help in 1-MCP treatment. This work was supported in part by Grants-in-Aid from the Ministry of Education, Science, Sports and Culture of Japan (18380037) and from the Japan Society for Promotion of Science (16-04536).
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