JXB Advance Access originally published online on December 21, 2006
Journal of Experimental Botany 2007 58(5):1047-1057; doi:10.1093/jxb/erl265
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RESEARCH PAPER |
Differential feedback regulation of ethylene biosynthesis in pulp and peel tissues of banana fruit
Faculty of Agriculture, Okayama University, Tsushima, Okayama, 700-8530 Japan
* To whom correspondence should be addressed. E-mail: ainaba{at}cc.okayama-u.ac.jp
Received 21 July 2006; Revised 8 November 2006 Accepted 10 November 2006
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Abstract |
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The feedback regulation of ethylene biosynthesis in banana [Musa sp. (AAA group, Cavendish subgroup) cv. Grand Nain] fruit was investigated in an attempt to clarify the opposite effect of 1-methylcyclopropene (1-MCP), an ethylene action inhibitor, before and after the onset of ripening. 1-MCP pre-treatment completely prevented the ripening-induced effect of propylene in pre-climacteric banana fruit, whereas treatment after the onset of ripening stimulated ethylene production. In pre-climacteric fruit, higher concentrations of propylene suppressed ethylene production more strongly, despite their earlier ethylene-inducing effect. Exposure of the fruit ripened by propylene to 1-MCP increased ethylene production concomitantly with an increase in 1-aminocyclopropane-1-carboxylate (ACC) synthase activity and ACC content, and prevented a transient decrease in MA-ACS1 transcripts in the pulp tissues. In contrast, in the peel of ripening fruit, 1-MCP prevented the increase in ethylene production and subsequently the ripening process by reduction of the increase in MA-ACS1 and MA-ACO1 transcripts and of ACC synthase and ACC oxidase activities. These results suggest that ethylene biosynthesis in ripening banana fruit may be controlled negatively in the pulp tissue and positively in the peel tissue. This differential regulation by ethylene in pulp and peel tissues was also observed for MA-PL, MA-Exp, and MA-MADS genes.
Key words: Banana fruit, enzyme activity, ethylene biosynthesis, fruit ripening, gene expression, pulp and peel tissue
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Introduction |
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Fruits have been classified as climacteric and non-climacteric on the basis of their patterns of respiration and ethylene production during maturation and ripening (Biale and Young, 1981). In climacteric fruits, it has been accepted that ethylene plays an important role in ripening where a massive production of ethylene commences at the onset of the respiratory climacteric rise, and that exogenously applied ethylene induces ripening and endogenous ethylene production. Banana, a typical climacteric fruit, has several unique characteristics. It exhibits a sudden increase followed by a rapid decrease in ethylene production, resulting in a sharp peak at the onset of ripening (Burg and Burg, 1965), different from other climacteric fruits where a gradual increase of ethylene production in parallel with a respiratory climacteric rise occurs. In addition, most of the climacteric fruits belong to dicotyledonous plants, while banana belongs to the monocotyledon group. Furthermore, the peel of banana fruit is a substantial tissue making up a large portion of the whole fruit, >20% of fresh weight, suggesting a possible important role in overall metabolism (Palmer, 1971).
In higher plants, ethylene is biosynthesized from methionine by a well-defined pathway in which 1-aminocyclopropane-1-carboxylate (ACC) synthase (ACS: EC 4.4.1.14 [EC] ) and ACC oxidase (ACO: EC 1.4.3) catalyse the reactions from S-adenosylmethionine (SAM) to ACC and from ACC to ethylene, respectively (Yang, 1987). It has been demonstrated that both ACS and ACO are encoded by multigene families in various plant organs (Kende, 1993; Zarembinski and Theologis, 1994; Fluhr and Mattoo, 1996). It is well known that ethylene biosynthesis is subject to both positive and negative feedback regulation (Yang and Hoffman, 1984; Kende, 1993). This opposite direction of feedback regulation in ethylene biosynthesis has been investigated at the level of transcriptional control in ACS and ACO genes. Examples of positive feedback regulation have been reported in several climacteric fruits such as tomato (Nakatsuka et al., 1998), passion fruit (Mita et al., 1998), pear (Lelièvre et al., 1997; Hiwasa et al., 2003), persimmon (Nakano et al., 2003), and kiwi fruit (Xu et al., 1998). The involvement of ACS genes in negative feedback regulation has been shown in non-climacteric fruits such as wounded winter squash (Nakajima et al., 1990; Kato et al., 2000) and chilling-injured citrus (Mullins et al., 1999). Even in climacteric fruits, it has been demonstrated that the basal level of system 1 ethylene produced in young tomato fruit is under a negative feedback control (Nakatsuka et al., 1998).
In spite of the above-mentioned unique features and commercial importance, research progress on ethylene biosynthesis in banana fruit has been hampered, probably due to its high concentrations of phenolic substances and starch. With the help of an advanced RNA extraction method using hot borate (Wan and Wilkins, 1994), three different cDNAs for ACS and one for ACO were isolated from banana fruit, and the expression characteristics of these genes were analysed during ripening (Liu et al., 1999). In this previous study, it was demonstrated that the sharp decline in ethylene production immediately following the initial burst of ethylene was caused by a sharp decline in ACO activity through limited availability of its cofactors. Furthermore, net ACS activity was detected in banana fruit by overcoming the interference of tannins (Liu et al., 2000) through the use of the polyethylene glycol (PEG)–acetone method (Badran and Jones, 1965). However, the feedback regulatory mechanism(s) involved in ethylene biosynthesis in banana fruit during ripening still remains unknown. Despite a sensitive induction of ripening by applied ethylene as described above, Golding et al. (1998) showed an involvement of a negative feedback regulatory mechanism in ethylene biosynthesis in banana fruit once ripening had commenced, which has led to the further investigation of this feature.
In the present study, the existence of negative and positive feedback regulation of ethylene biosynthesis is demonstrated in banana pulp and peel tissues, respectively.
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Materials and methods |
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Plant materials and treatments
Pre-climacteric bananas [Musa sp. (AAA group, Cavendish subgroup) cv. Grand Nain] fruit imported from the Philippines were supplied by a local importer. Each banana hand was separated into individual fingers. Fingers were treated or not with 20–40 µl l–1 1-methylcyclopropene (1-MCP) for 6 h, followed by treatment with 500 µl l–1 propylene for 18 h, and then ripened at 22 °C. Ethylene production was monitored at appropriate intervals and then pulp tissues were frozen in liquid nitrogen for the extraction of total RNA. In another experiment, fingers were treated with 500 µl l–1 propylene for 18 h, followed by treatment or not with 20–40 µl l–1 1-MCP for 6 h on day 1 or day 2 during ripening at 22 °C. Ethylene production from intact fingers was monitored every day, and then fingers were separated into pulp and peel tissues. The pulp tissues adhering to the peel were removed as much as possible using a razor blade. Ethylene production from each tissue was determined after 1 h incubation in air to remove pre-existing ethylene in the tissues. After ethylene determination, the pulp and peel tissues were frozen in liquid nitrogen for the extraction of total RNA, ACC, ACS, and ACO. To examine any inhibitory effect of propylene on the ethylene production, fingers were continuously treated with 50, 500, and 5000 µl l–1 of propylene, and ethylene production was monitored. In each experiment, fingers from the same hand were used as a sample group to avoid variation in ripening behaviour of fingers among different hands. 1-MCP synthesis and treatment were carried out as described previously (Nakatsuka et al., 1998).
Measurement of respiration rate, peel colour, and flesh firmness
The respiration rate was determined as carbon dioxide production measured by enclosing fruit samples in an airtight container for 1 h at 22 °C, withdrawing 1 ml of the headspace gas, and injecting it into a gas chromatograph (model GC-3BT, Shimadzu, Kyoto, Japan) equipped with a thermal conductivity detector and a Porapack Q column. Skin colour was measured at three regions of the fruit surface and expressed as hue angle (H°) calculated from the determined values with a colour difference meter (model 1000DP, Nippon Denshyoku Kogyo, Tokyo, Japan). Flesh firmness was measured at three regions of the peeled flesh using a penetrometer (model STM-T-50P, Toyo Baldwin, Tokyo, Japan) fitted with an 8 mm plunger and expressed in Newtons (N).
Extraction of ACS and ACO, and determination of their activities
ACS and ACO were extracted according to the method established previously (Liu et al., 2000). A 5 g aliquot of frozen pulp and peel tissues was homogenized in a Waring blender (Model AM-3, Nissei, Tokyo, Japan) for 3 min on ice at maximum speed in 25 ml of the extraction buffer with added PEG, consisting of 100 mM K-phosphate (pH 8.5), 5 mM dithiothreitol (DTT), 5 µM pyridoxal-5'-phosphate, 10% (v/v) glycerol, and 1% (w/v) PEG (average mol. wt. 8000 Da, Sigma, St Louis, MO, USA). ACS and ACO were extracted as described previously (Liu et al., 1999, 2000). ACS activity was assayed by incubating 1 ml of enzyme preparation at 30 °C for 20 min with 200 µM SAM and 100 mM K-phosphate buffer (pH 9) in a total volume of 1.5 ml. The reaction was terminated with 0.1 ml of 50 mM HgCl2. The amount of ACC formed was determined by the method of Lizada and Yang (1979). ACO activity was assayed by incubating 0.5 ml of the enzyme preparation with 100 mM K-phosphate buffer (pH 7.5), 10% (v/v) glycerol, 1 mM ACC, 30 mM sodium ascorbate, 0.1 mM FeSO4, and 20 mM NaHCO3 at 30 °C for 20 min, and the ethylene produced was determined by gas chromatography as described below. Both enzyme activities were expressed as the amount of ethylene (in nmol) produced g–1 FW h–1.
Measurement of ethylene production, ACC content, and in vivo ACO activity
Ethylene production was measured by enclosing fruit samples in an airtight container for 1 h at 22 °C, withdrawing 1 ml of the headspace gas, and injecting it into a gas chromatograph (model GC-4CMPF, Shimadzu) fitted with a flame ionization detector and an activated alumina column. ACC content was measured by the method of Lizada and Yang (1979), with 80% (v/v) ethanol extracts from frozen fruit samples. For the measurement of in vivo ACO activity, slices of 1 mm thickness (
1 g) of pulp, or slices of 1 g weight of peel were put into 40 ml Erlenmeyer flasks containing 2 ml of incubation buffer consisting of 1 mM ACC and 100 mM TRIS-HCl (pH 7.5). The flasks were incubated at 30 °C for 1 h and the ethylene formed was determined as described above. The activity was expressed as ethylene (in nmol) produced g–1 FW h–1.
RNA isolation, cloning, and sequencing
The methods of RNA extraction, cloning, and DNA sequencing were the same as described previously (Liu et al., 1999). The first-strand cDNAs synthesized from poly(A)+ RNA isolated from ripe banana pulp were used as templates for reverse transcription-polymerase chain reaction (RT-PCR). The cDNA for MA-ACS4 was cloned using the degenerate primers for ACS designed in a previous report (Liu et al., 1999). Specific primers for MA-ACO2, MA-PL1, MA-PL2, MA-Exp1, and MA-Exp2 were designed from their nucleotide sequences registered on the database (X95599
[GenBank]
, AF206319
[GenBank]
, AF206320
[GenBank]
, AY083168
[GenBank]
, and AF539540
[GenBank]
), as were specific primers for three banana MADS-box genes, MA-MADS1, MA-MADS2, and MA-MADS3 (AY941798
[GenBank]
, AY941799
[GenBank]
, and AY941800
[GenBank]
).
RT-PCRs consisted of 30 cycles of 94 °C for 1 min, 55 °C for 2 min, and 72 °C for 3 min. The amplified cDNAs were ligated into either pUC118 (Takara, Kyoto, Japan) or pGEM-T Easy (Invitrogen, Carlsbad, CA, USA) vectors. To determine the longer nucleotide sequences for MA-MADS cDNAs, RACE-PCR (rapid amplification of cDNA ends) was performed using a Marathon cDNA amplification kit (CLONTECH, Palo Alto, CA, USA) according to the manufacturer's protocol. The nucleotide sequences of the cDNA inserts were determined using a DNA sequencer (model DSQ-1000, Shimadzu).
Northern blot analysis
The mRNAs isolated from banana pulp and peel tissues were separated by electrophoresis on 1% (w/v) agarose gels containing 0.66 M formaldehyde, blotted onto nylon membranes (Hybond N, GE Healthcare Bio-Sciences, Piscataway, NJ, USA), and fixed by a UV cross-linker (GE Healthcare Bio-Sciences). The membranes were hybridized with 32P-labelled cDNA probes obtained from the RT-PCR products mentioned above and washed under the same conditions described previously (Liu et al., 1999). The membranes were subsequently exposed to an imaging plate (Fuji Photo Film, Tokyo, Japan) at room temperature. Equal reactivity and amount of RNA in all samples were verified by hybridization with 32P-labelled MA-Actin cloned previously (Liu et al., 1999). For analysis of the pectate lyase and expansin genes mentioned above, gene-specific digoxigenin-labelled probes prepared with a PCR DIG Probe synthesis kit (Roche Diagnostics, Mannheim, Germany) were used and signals were detected by chemiluminescence using CDS-Star (Roche Diagnostics) as described previously (Hiwasa et al., 2003).
Quantification of MA-MADS transcripts by real-time PCR
cDNA was synthesized using SuperScript II RT (Invitrogen) with oligo(dT) primers. Accumulation levels of MA-MADS2 transcripts were analysed by a real-time PCR method, with an iCycler Real-Time PCR System (Bio-Rad, Hercules, CA, USA) monitoring the amplification with the SYBR-Green I dye (Applied Biosystems, Foster City, CA, USA). MA-Actin cDNA was used as an internal constitutively expressed control. Sequences of primers used were: 5'-CCAGATTGAGGTAGAAGGTGCCACC-3' and 5'-TGGGTTCCTGGGAATCGCAGCATCA-3' for MA-MADS2, and 5'-TGCTAGCGGACGTACCACAGGTATCGTG-3' and 5'-GAATCTTCATGAGGGAATCTGTCAGGTC-3' for MA-Actin.
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Cloning of cDNAs
In the present study, one more cDNA fragment for both ACS (MA-ACS4) and ACO (MA-ACO2) was cloned from the pulp of banana fruit in addition to the genes cloned previously (MA-ACS1, MA-ACS2, MA-ACS3, and MA-ACO1) (Liu et al., 1999). The MA-ACS4 gene showed a high sequence identity at the nucleotide level to MA-ACS2 and MA-ACS3 of 82% and 84%, respectively, and a relatively low identity of 63% to MA-ACS1.
Effect of 1-MCP pre-treatment on induction of ethylene biosynthesis by propylene
In pre-climacteric banana fruit treated with propylene for 18 h, ethylene production was induced within 1 d. This propylene-induced ethylene production was completely prevented by pre-treatment with 1-MCP (Fig. 1A). Fruit pre-treated with 1-MCP began to ripen from day 20, with ethylene production reaching a much higher level than that in propylene-treated control fruit. Figure 1B shows the northern analysis for MA-ACS1, MA-ACS4, MA-ACO1, and MA-ACO2 genes related to ripening ethylene biosynthesis in banana fruit, using mRNAs extracted from the pulp tissue at the corresponding date indicated in Fig. 1A. Accumulation of MA-ACS1 mRNA was undetectable at the pre-climacteric stage, but was induced strongly by propylene treatment. The abundance of MA-ACO1 transcript was already detectable at the pre-climacteric stage and significantly enhanced by propylene treatment. These induction or enhancement effects of propylene on the accumulation of these mRNAs relating to ethylene biosynthesis were completely inhibited by 1-MCP until ripening commenced. MA-ACS4 was expressed constitutively throughout the ripening process, irrespective of both propylene and 1-MCP treatments. The MA-ACO2 gene showed a similar, but much weaker, expression pattern compared with the MA-ACO1 gene. Thus the roles of MA-ACS4 and MA-ACO2 in ethylene biosynthesis seemed to be less important in banana fruit ripening, and further analysis of these genes was not performed.
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Effect of 1-MCP treatment after onset of ripening on ethylene production and inhibition of ethylene induction by a high concentration of propylene
Figure 2A shows the effect of 1-MCP application to the fruit ripened previously by propylene on subsequent ethylene production during ripening. 1-MCP application 1 d after propylene treatment not only prevented progress of the ethylene climacteric rise but also enhanced the rate of ethylene production. However, other signs of ripening such as the increase in respiration rate, decrease in flesh firmness, and changes in skin colour were inhibited in 1-MCP-treated fruit (Fig. 2B–D). These results suggest that 1-MCP could be active even in ripening banana fruit producing ethylene with respect to prevention of its action. Therefore, ethylene biosynthesis in ripening banana fruit may be regulated in an autoinhibitory manner or independently of ethylene, although it is inducible by propylene at the pre-climacteric stage. To confirm this hypothesis, banana fruit were treated with different concentrations of propylene (Fig. 3). Higher concentrations of propylene induced ethylene earlier, but production rates at the peak were lower, suggesting an involvement of autoinhibitory ethylene biosynthesis in ripening banana fruit contrary to the common belief that it is a typical climacteric fruit. To investigate this observation, ethylene production was determined in the pulp and peel tissues separately in the fruit treated with 1-MCP because commercially utilized banana has a thick peel accounting for about one-fifth of the total weight. Interestingly, ethylene biosynthesis was regulated in the opposite direction in the pulp and peel (Fig. 4). In the pulp, 1-MCP treatment during progress of the ethylene climacteric rise greatly enhanced subsequent ethylene production 2–3-fold throughout ripening (Fig. 4A). In contrast, in the peel, an increase in ethylene production accompanying the ripening process was almost completely suppressed by 1-MCP treatment (Fig. 4B). In propylene-treated control fruit, ethylene production in peel was detectable from day 2 and then increased gradually towards the late ripening stages, followed by a slight decrease, different from that in pulp with a peak on day 2.
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Comparison of internal feedback regulation of ethylene biosynthesis in pulp and peel tissues
Figure 5 shows the changes in ACS activity, ACC content, in vivo and in vitro ACO activities, and northern analysis for the pulp used in the determination of ethylene production (Fig. 4A). In propylene-treated control pulp, ACS activity peaked on day 2 and decreased thereafter, while the ACC content gradually increased during ripening with a slight decline at later ripening stages (Fig. 5A, B). These increases in ACS activity and ACC content associated with the ripening process were greatly enhanced by treatment of fruit with 1-MCP, suggesting an involvement of a negative feedback regulation of ethylene biosynthesis in the pulp at the ACS level. In propylene-treated control pulp, in vivo ACO activity peaked on day 1 and thereafter declined to the initial level, while in vitro ACO activity steadily increased toward day 4 (Fig. 5C, D). 1-MCP treatment did not affect either in vivo or in vitro ACO activities in the pulp. Figure 5E shows the northern analysis for MA-ACS1, MA-ACS2, and MA-ACO1 mRNAs in the pulp. Accumulation of MA-ACS1 mRNA was undetectable at the pre-climacteric stage and strongly induced on day 1 with commencement of ripening induced by propylene treatment. The increased abundance of this gene with ripening weakened transiently on day 3, followed by stimulation again thereafter. 1-MCP inhibited this transient weakening of MA-ACS1 transcripts and enforced the later stimulation slightly. Signals for MA-ACS2, a wound-inducible gene (Liu et al., 1999), were not detected in all samples. 1-MCP treatment had no effect on the constitutive expression of the MA-ACO1 gene in pulp once ripening had commenced.
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In the peel, 1-MCP had the opposite effects to those observed in the pulp, as shown in Fig. 6. In peel of the propylene-treated control fruit, ACS activity (Fig. 6A) and ACC content (Fig. 6B), and in vivo and in vitro ACO activities (Fig. 6C, D) increased as ripening progressed. These increases in ACS and ACO activities with ripening were strongly suppressed by 1-MCP treatment. The suppressive effects of 1-MCP were observed in expression of the MA-ACS1 and MA-ACO1 genes (Fig. 6E). Accumulation of MA-ACS1 and MA-ACO1 mRNAs in the propylene-treated control peel was undetectable at the pre-climacteric stage and slightly induced on day 1, followed by a strong level of detection. The enhanced expression of these genes with ripening was completely suppressed by 1-MCP. Expression of MA-ACS1 and MA-ACO1 genes in 1-MCP-treated peel tissue was almost eliminated and decreased to the basal level, respectively, suggesting that a positive feedback regulation may be involved in ethylene biosynthesis in banana peel. No signals for MA-ACS2 transcripts were detected in any peel tissues tested.
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Effect of 1-MCP treatment after onset of ripening on expression of genes related to fruit softening and of MADS-box genes
To clarify the existence of different regulatory mechanism(s) between pulp and peel tissues, the expression of MA-PL and MA-Exp genes, which is known to be ethylene dependent, and MADS-box genes, which have been suggested to be ripening requirement factor(s) upstream of ethylene, were analysed. As shown in Fig. 7, both the pectate lyase and expansin genes were expressed from day 2 until the end of the experiment in both pulp and peel tissues. 1-MCP had almost no effect on the expression of these genes in the pulp, whereas expression was strongly inhibited in the peel tissues. Among the three MA-MADS genes, MA-MADS2 showed the greatest abundance of mRNA in the peel and the lowest in the pulp (Fig. 8). 1-MCP greatly suppressed the expression of this gene in the peel and slightly in the pulp. MA-MADS1 and MA-MADS3 genes were almost constitutively expressed in the peel and pulp during ripening irrespective of 1-MCP treatment (data not shown).
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Discussion |
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Unlike most climacteric fruits, banana has a characteristic feature of a sharp rise and fall in the rate of ethylene production at the onset of natural ripening. In a previous report (Liu et al., 1999), a possible regulatory mechanism of this sharp peak of ethylene production was demonstrated, as follows: (i) an increase in the abundance of MA-ACS1 mRNA in the flesh is the first step of ethylene induction at the onset of the climacteric rise; (ii) the increased content of ACC in the flesh induces a rapid increase of climacteric ethylene whereby ACO is activated with the enhanced accumulation of MA-ACO1 mRNA once ripening commences; and (iii) ACO activity decreases rapidly through limitation of its cofactors, i.e. a decline in the ascorbate and iron contents. At the same time as this previous study, Golding et al. (1998) showed interesting features of ethylene production in banana fruit, demonstrating that 1-MCP eliminated the ripening induction effect of propylene at the pre-climacteric stage whereas it had no effect on ethylene production once ripening had commenced. These results led to the suggestion that some unique regulatory mechanism(s) may be involved in ethylene biosynthesis in banana fruit. Furthermore, ACS activity could be determined in banana fruit (Liu et al., 2000) by the use of the PEG–acetone method described by Badran and Jones (1965). Although the determination of ACS activity is essential to understand the regulatory mechanism of ethylene biosynthesis, no reports have been found yet because of the difficulty in its extraction from banana fruit probably due to a high concentration of tannins. With this background information and to expand on the results reported by Golding et al. (1998), the characteristics of ethylene production associated with ripening in banana fruit were investigated further at the levels of enzyme activity and gene expression.
First, one novel cDNA fragment was cloned for ACS in addition to the fragments cloned previously (Liu et al., 1999). Banana genomic Southern analysis in this previous study (Liu et al., 1999) suggested that at least one more gene homologous to MA-ACS2 and MA-ACS3, a wound-inducible gene and a gene not expressed in the pulp, respectively, could exist in the same subfamily. The MA-ACS4 gene cloned in this study indeed showed a high sequence similarity to these two genes.
It has been accepted that ethylene biosynthesis in climacteric fruits may be regulated by a positive feedback mechanism whereby exogenously applied ethylene induces endogenous ethylene production resulting in commencement of ripening in this type of fruit. This concept has been clearly supported in various climacteric fruits, including apple (Fan et al., 1999), pear (Lelièvre et al., 1997; Hiwasa et al., 2003), and tomato (Nakatsuka et al., 1998), where 1-MCP treatment could delay or eliminate the accumulation of mRNA for genes related to ethylene biosynthetic enzymes when applied to the fruit at pre-climacteric or ripening stages, respectively. Banana has been widely known as a typical climacteric fruit because ripening is induced by exogenous ethylene treatment. This induction of ripening by exogenous ethylene was inhibited by 1-MCP pre-treatment at the pre-climacteric stage (Golding et al., 1998; Jiang et al., 1999; Harris et al., 2000; Pathak et al., 2003). However, an interesting observation was made by Golding et al. (1998) where it was shown that 1-MCP had no suppressive effect on endogenous ethylene production in banana fruit once ripening has commenced.
In the present study, it was re-confirmed that 1-MCP had a strong preventive effect of ripening induced by propylene with respect to delay of expression of the genes related to ethylene production in pre-climacteric banana fruit when applied before propylene treatment (Fig. 1). However, 1-MCP did not inhibit ethylene production, but rather had a stimulative effect when applied after the onset of ripening, although other ripening progress was inhibited by 1-MCP (Fig. 2). These results, together with those of Golding et al. (1998), suggest that ethylene biosynthesis in banana fruit is regulated autocatalytically at least during the pre-climacteric period, but is independent of ethylene or under a negative feedback regulation mechanism at the ripening stage. To confirm this hypothesis, pre-climacteric fruit were treated with different concentrations of propylene. It has been widely accepted that propylene has an action on ethylene at one-hundredth the concentration and has been used to determine endogenous ethylene induction under exogenous ethylene treatment in various plant organs. In this study, it was found that higher concentrations suppressed ethylene production more strongly than lower concentrations despite their earlier ethylene induction effect (Fig. 3). This induced ethylene may be derived from the pulp because peel tissues did not produce ethylene until day 2 (Fig. 4B). Thus, autoinhibitory regulation of ethylene biosynthesis may be operative in banana fruit at least in the pulp. A similar proposal based on suppression of endogenous ethylene production by exogenous ethylene has previously been shown in banana fruit (Vendrell and MacGlasson, 1971; McMurchie et al., 1972; Dominguez and Vendrell, 1994). These observations may introduce a new concept that, in banana fruit, exogenous ethylene does not induce endogenous ethylene production but induces only ripening, and the ripening induces endogenous ethylene production which seems to be under a negative feedback regulatory mechanism.
To understand this complex regulation system further, the fruit were separated into pulp and peel tissues because banana fruit had a thick peel tissue. Interestingly, 1-MCP applied to banana fruit 1 d after onset of the climacteric rise enhanced ethylene production in the pulp greatly while suppressing it almost completely in the peel, suggesting the existence of different feedback regulatory mechanisms of ethylene biosynthesis in these fruit tissues (Fig. 4). This was supported by the activity of ethylene biosynthesis and related gene expression in both tissues.
In pulp of the fruit treated with propylene, the accumulation of MA-ACS1 mRNA was induced on day 1 and declined toward day 3, and was well correlated with changes in ACS activity and ACC content (Fig. 5). On day 3, signals for the MA-ACS1 transcript declined transiently to a very low level while 1-MCP suppressed this. This transient decline on day 3 was also observed in the banana pulp treated with ethylene, as shown previously (Liu et al., 1999), suggesting a reflection of negative feedback regulation. The negative feedback regulatory mechanism of ACS genes has been reported to be involved in wound-induced ethylene production. In this study, fruit were separated into pulp and peel tissues, and ethylene produced from each tissue may contain some wound-induced ethylene. However, this concern was excluded from the results because no signals for MA-ACS2 transcripts were detected in any of the tissues examined. The MA-ACS2 gene has been shown to be expressed in response to wounding stimuli only (Liu et al., 1999). Interestingly, the ACS gene from citrus (Mullins et al., 1999), the CMe-ACS gene from winter squash (Nakajima et al., 1990; Kato et al., 2000), Le-ACS1A and Le-ACS6 from tomato (Tatsuki and Mori, 1999), and Ps-ACS2 from etiolated pea seedling (Peck and Kende, 1998), all exhibited a transient expression correlating with a temporal increase in ethylene production and/or ACS activity. In vivo and in vitro ACO activity and its gene expression in the pulp of the fruit treated with propylene in this study showed the same trend observed previously (Liu et al., 1998). A rapid rise and fall in the in vivo ACO activity in propylene-treated fruit may be caused by limited availability of its cofactors, iron and ascorbate. This was also supported in this study by the fact that in vitro ACO activity determined with the cofactors increased steadily until the fully ripe stage. Expression of MA-ACO1 mRNA was greatly increased by propylene treatment and no change was observed throughout ripening. 1-MCP had no effect on the enzyme activity and gene expression of ACO in the pulp. These results suggest that ethylene biosynthesis in banana pulp may be regulated under negative feedback at the ACS level.
In contrast to the pulp, 1-MCP greatly suppressed both ACS and ACO activities in the peel, with a strong effect on their gene expression (Fig. 6), suggesting an involvement of a positive feedback regulatory mechanism in ethylene biosynthesis in the peel tissue. In the peel, ethylene production began to increase from day 2 with increases in ACS and in vitro and in vivo ACO activities toward the fully ripe stage. Studies on ethylene biosynthesis in banana fruit during the past three decades have suggested that the peel hardly produces any ethylene and the production in the peel may be from the ethylene produced in the pulp (Vendrell and MacGlasson, 1971; Dominguez and Vendrell, 1993; Clendennen and May, 1997). Therefore, it was felt that sufficient endogenous ethylene for a response to 1-MCP had not yet been induced in the peel at day 1. To eliminate this concern and to determine the expression pattern of ethylene-dependent genes, fruit were treated with 1-MCP on day 2 and the northern blot analysis was repeated with the pectate lyase and expansin genes in addition to MA-ACS1. Expression of these genes is well known to be under a positive feedback regulation by ethylene in most climacteric fruits. 1-MCP treatment had almost no effect on the propylene-induced expression of these genes in the pulp, whereas the expression in the peel was almost eliminated by the treatment with 1-MCP (Fig. 7). These results strongly support the hypothesis that a different feedback regulation operates in the pulp and peel tissues of banana fruit.
It is well known that all ripening phenomena are inhibited in the rin tomato mutant. Vrebalov et al. (2002) revealed that this ripening-inhibited feature of rin tomato derived from lesion of the LeMADS-RIN gene by positional cloning and showed that transgene integration of this gene from wild-type tomato into the rin mutant showed recovery of ripening phenomena. From these observations, they suggest that LeMADS-RIN, one of the MADS-box family genes, is required for fruit ripening upstream of ethylene. Based on these findings, the expression of banana MADS-box genes was examined (Fig. 8). Among the three genes, only MA-MADS2 showed a large difference in mRNA accumulation between the peel and pulp tissues. MA-MADS2 mRNA accumulated 100-fold more in the peel than in the pulp, and its accumulation in the peel was lowered by 1-MCP treatment. Therefore, MA-MADS2 may relate to the opposite direction of feedback regulation by ethylene in peel and pulp tissues.
In conclusion, ethylene biosynthesis in banana fruit was regulated in opposite directions in the pulp and peel. Ethylene biosynthesis may be under a negative feedback regulation mechanism in the pulp whereas a positive feedback system may operate in the peel. This difference may be derived from the different regulatory mechanisms at the transcriptional level of MA-ACS1 and MA-ACO1 genes in the pulp and peel tissues. Furthermore, based on the different effects of 1-MCP before and after onset of ripening, at least in banana fruit, it seems that exogenous ethylene does not induce endogenous ethylene production but induces only ripening, which in turn induces massive endogenous ethylene production that is regulated under a negative feedback mechanism.
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Acknowledgements |
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We thank Dr Francis M Mathooko (Jomo Kenyatta University of Agriculture and Technology, Kenya) for his careful reading of the manuscript. This work was supported in part by Grants-in-Aid for Scientific Research (grant no. 14360023 to AI) from the Ministry of Education, Science, Sports, and Culture of Japan. The sequence of MA-ACS4 of bananas has been submitted to DDJB under accession number AB266314.
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Footnotes |
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Present address: Procter & Gamble Far East, Inc., Higashinada-ku, Kobe, 658-0032 Japan. 
Present address: Research Institute for Biological Sciences, Okayama, Kibichuo-cho, Okayama, 716-1241 Japan.
Present address: Department of Horticulture, South China Agricultural University, Guangzhou 510642, China.
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Abbreviations |
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ACC, 1-aminocyclopropane-1-carboxylate, ACO, ACC oxidase; ACS, ACC synthase; 1-MCP, 1-methylcyclopropene; PEG, polyethylene glycol; RT-PCT, reverse transcription-polymerase chain reaction.
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