Journal of Experimental Botany, Vol. 54, No. 381, pp. 309-316,
January 2, 2003
© 2003 Oxford University Press
Malate synthase gene expression during fruit ripening of Cavendish banana (Musa acuminata cv. Williams)
Received 25 June 2002; Accepted 12 September 2002
Plant Genetic Engineering Laboratory, Department of Biological Sciences, Faculty of Science, National University of Singapore, 10 Kent Ridge Crescent, Singapore 119260, Republic of Singapore
1 To whom correspondence should be addressed. Fax: +65 6779 2486. E-mail: dbspuaec{at}nus.edu.sg
2 Present address: Department of Plant Biology, Cornell University, Ithaca, New York 14853, USA.
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Abstract |
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Malate synthase (MS) is a key enzyme responsible for malic acid synthesis in the glyoxylate cycle, which functions to convert stored lipids to carbohydrates, by catalysing the glyoxylate condensation reaction with acetyl-CoA in the peroxisome. In this study, the cloning of an MS cDNA, designated MaMS-1, from the banana fruit is reported. MaMS-1 was 1801 bp in length encoding a single polypeptide of 556 amino acid residues. Sequence analysis revealed that MaMS-1 possessed the conserved catalytic domain and a putative peroxisomal targeting signal SK(I/L) at the carboxyl terminal. MaMS-1 also shared an extensive sequence homology (79–81.3%) with other plant MS homologues. Southern analysis indicated that MS might be present as multiple members in the banana genome. In Northern analysis, MaMS-1 was expressed specifically in ripening fruit tissue and transcripts were not detected in other organs such as roots, pseudostem, leaves, ovary, male flower, and in fruit at different stages of development. However, the transcript abundance in fruit was affected by stage of ripening, during which transcript was barely detectable at the early stage of ripening (FG and TY), but the level increased markedly in MG and in other fruits at advanced ripening stages. Furthermore, MaMS-1 expression in FG fruit could be stimulated by treatment with 1 µl l–1 exogenous ethylene, but the stimulatory effect was abolished by the application of an ethylene inhibitor, norbornadiene. Results of this study clearly show that MS expression in banana fruit is temporally regulated during ripening and is ethylene-inducible.
Key words: Banana, ethylene, fruit ripening, gene expression, malate synthase, Musa acuminata.
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Introduction |
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The enzyme malate synthase (MS, EC 4.1.3.2) is responsible for malate synthesis by catalysing the Claisen condensation of glyoxylate with acetyl-CoA in the glyoxysome. Both MS and isocitrate lyase (ICL) are involved in the glyoxylate cycle that is important to the maintenance of gluconeogenesis. The process of gluconeogenesis is initiated by conversion of stored lipids to carbohydrates, which is active in several plant systems. In photosynthetically incompetent seedlings, where ICL and MS activities are high, lipids have been utilized as the carbon source for growth (Escher and Widmer, 1997). The gluconeogenic role of the glyoxylate cycle is also important for seed germination and post-germinative growth of oilseed plants, where oils are the main seed storage reserves (Eastmond and Graham, 2001). Apart from gluconeogenesis, the glyoxylate cycle also plays an anaplerotic role to replenish the intermediates, for example, succinate, to the TCA cycle, which is the major cellular machinery for oxidative metabolism and energy production (Graham et al., 1994; Eastmond and Graham, 2001).
ICL and MS have also been shown to play a role in various plant processes during growth and development. These include pollen development (Zhang et al., 1994) and the transition from late embryogeny to germination (Ettinger and Harada, 1990) of oilseed rape (Brassica napus), and senescence of several plant species such as pumpkin (Cucurbita pepo) (De Bellis and Nishimura, 1991; Pistelli et al., 1996), barley (Hordeum vulgare) (Gut and Matile, 1988), wheat (Triticum durum), and rice (Oryza sativa) (Pistelli et al., 1991). The importance of the glyoxylate cycle has been demonstrated in the ICL-defective Arabidopsis thaliana mutant that lacked the glyoxylate cycle (Eastmond et al., 2000). The survival and recovery of this mutant were severely affected after growing in the dark. Changes in gene expression and/or activity of glyoxylate cycle enzymes in plants have been associated with environmental cues. In tomato (Lycopersicon esculentum), the activity of MS and ICL in seedlings grown in the dark increased, but decreased after illumination (Nieri et al., 1997). Starvation by removal of sugars from the growing environment has been shown to up-regulate accumulation of MS transcripts in cucumber (Cucumis sativus) cell culture (Graham et al., 1994) and increased enzyme activity in maize (Zea mays) root tips (Dieuaide et al., 1992). Furthermore, the stomatal function has also been related to malate accumulation in guard cells (Laporte et al., 2002). Although the physiological role of MS in various aspects of plant growth and development has been well documented, information regarding fruit ripening in relation to MS has been limited.
Fruit ripening is a complex process that involves drastic changes in various physiological and biochemical events. These events include chlorophyll breakdown, increased starch degradation and sugar synthesis, and fruit softening. The ripe fruit also develops unique flavours that result, at least in part, from increased synthesis of organic acids (Grierson and Kader, 1986). In banana (Musa sp.), both citrate and malate are the predominant organic acids in the green fruit (John and Marchal, 1995). The malate content in banana fruit has also been shown to increase markedly as ripening progressed (Satyan and Parwardhan, 1984). On the other hand, preclimacteric banana fruit subject to gamma irradiation showed an increase in MS and ICL activities and the soluble sugar content, but succinate dehydrogenase, a key enzyme of the TCA cycle, was inhibited (Surendranathan and Nair, 1976). However, the TCA cycle was not affected, indicating that the functional glyoxylate cycle may play an anaplerotic and/or gluconeogenetic role in the fruit.
As part of a long-term interest to understand the molecular basis of fruit ripening in banana, this study is a report on the isolation of a cDNA encoding MS from ripening banana fruit. The MS cDNA was characterized by spatial and temporal expression in various organs and during fruit ripening. Results provide the evidence showing, for the first time, that ethylene may play a regulatory role in MS expression.
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Materials and methods |
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Plant materials
Cavendish banana (Musa acuminata cv. Williams) plants grown in the departmental garden were used. Fruits at 20–100 d after flower shooting (DAS) were harvested for developmental study. For the ripening study, fruits harvested at the mature green stage (100 DAS) were allowed to ripen in the air at room temperature (24±2 °C). The degree of ripening was classified into seven stages, according to Stover and Simmonds (1987). These stages were full green (FG), trace yellow (TY), more green than yellow (MG), more yellow than green (MY), green tip (GT), full yellow (FY), and yellow flecked with brown spots (YB).
Cloning and sequence analysis of MS gene
One expressed sequence tag (EST) clone, designated MaMS, isolated from the cDNA library constructed from ripening fruit (Pua et al., 2000) was putatively identified as part of the MS gene by partial sequencing. The complete nucleotide sequence of MaMS was subsequently determined by automated sequencing using the ABI PRISMTM Big DyeTM Terminator Cycle Sequencing Ready Reaction Kit and the ABI PRISMTM 377 DNA sequencer (Applied Biosystems/Perkin Elmer, USA).
The nucleotide and deduced amino acid sequences were determined using the BLAST sequence homology search (Altschul et al., 1997) provided by the National Center for Biotechnology Information.
Genomic DNA isolation and Southern analysis
Genomic DNA was isolated from developing leaves of banana using the cetyl triethylammonium bromide method (Doyle and Doyle, 1987) with slight modifications (Pua et al., 2000). For Southern analysis, 15 µg of genomic DNA was restricted overnight with various endonucleases AccI, AflII, BglII, EcoRI, EcoRV, and XbaI. The DNA was separated by gel electrophoresis, depurined, and transferred overnight onto a positively charged nylon membrane by capillary action as previously described (Pua et al., 2000). The membrane was crosslinked with UV radiation, prehybridized at 42 °C in digoxigenin (DIG) Easy Hyb buffer (Roche Molecular Biochemicals, Germany), and hybridized overnight in the same buffer containing the probe, which was prepared from a partial MS cDNA, MaMS, isolated in this study. The probe was labelled with DIG and detected using the luminescent detection kit according to manufacturer’s instructions (Roche Molecular Biochemicals).
RNA isolation and Northern analysis
Total RNA was isolated from the various organs of banana as previously described (Pua et al., 2000). For Northern analysis, 20 µg of total RNA was denatured in glyoxal and dimethyl sulphoxide, separated by gel electrophoresis, and transferred to a positively charged nylon membrane. After crosslinking, the membrane was treated and probed with DIG-labelled MaMS as described in Southern analysis.
Treatment of tissues with exogenous ethylene
The effect of ethylene on MS expression in fruit was investigated by treatment of FG fruit for 24 h with different concentrations of ethylene (0, 1 and 20 µl l–1) alone or in combination with 2 or 20 ml l–1 norbornadiene (NBD) in a 9.0 l desiccator sealed with an air-tight rubber stopper as previously described (Pua et al., 2000). For the control treatment (0 µl l–1 ethylene), only air was introduced. After treatment, the fruit pulp and peel were separated, frozen in liquid nitrogen, and stored at –80 °C until use.
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Results |
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From a banana fruit cDNA library, an EST MaMS that was highly homologous to plant MS genes has previously been isolated. MaMS was further sequenced in this study. Sequence analysis revealed that MaMS was 1680 bp in length encoding a single polypeptide of 519 amino acid residues, but the translation start site was absent in MaMS. The cDNA was also shorter than maize (Paek et al., 1995) and cucumber MS (Graham et al., 1989) by 38 and 49 residues, respectively, indicating that MaMS may not encode the full length MS in banana. To obtain the full length cDNA, the forward T3 universal primer from the pBluescript vector and 20-mer reversed gene-specific primer (5'-GTCAGCCATGCCGACTTTGG-3') from MaMS were synthesized. After PCR using the cDNA library as template, a 462 bp DNA fragment was amplified and sequenced. Sequence analysis revealed that the amplified fragment encoded a polypeptide of 121 amino acid residues, including the putative translation start site, and a short (10 bp) 5'-untranslated region, and a region of 84 residues at the C-terminus that was completely mapped to the 5'-end of MaMS. This result indicated that the amplified fragment might have carried the missing 5' sequence of MaMS. The joining of the amplified fragment, excluding the overlapping region, with MaMS yielding the full-length MS cDNA, designated as MaMS-1 (Genbank Accession No. AF321286). MaMS-1 was 1801 bp in length encoding a protein of 556 amino acid residues (Fig. 1). The size was comparable to that of a maize MS that possessed 568 residues (Paek et al., 1995).
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. Arrow represents the downstream reverse gene-specific primer used for DNA amplification (the upstream forward T3 universal primer is not shown). An overlapping region between the PCR-amplified fragment and the EST is double-underlined. The putative polyadenylation signal at the 3'-UTR is boxed.MaMS-1 shared a high degree of sequence similarity with MS of other plant species, showing 78% homology with cucumber (Graham et al., 1989) and cotton (Gossypium hirsutum) (Turley et al., 1990), 77% with rapeseed (J04468), 76% with soybean (Glycine max) (Guex et al., 1995) and pumpkin (X56948), 74% with maize (Paek et al., 1995), and 69% with radish (Raphanus sativus) (X78852) (Fig. 2). Apart from high sequence similarity, MaMS also possessed the putative catalytic domain and the tripeptidyl glyoxysomal targeting signal of SK(I/L) at the carboxyl terminus, which are highly conserved in MS.
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Southern analysis was conducted to determine the relative copy number of the MS gene in the banana genome. Genomic DNA was restricted with BglII, EcoRI, EcoRV, and XbaI that did not cut within the MaMS probe. Results showed that MaMS strongly hybridized to two DNA bands with BglII digest, three bands with EcoRI and EcoRV digests, and four bands with XbaI digest (Fig. 3), suggesting that MS in banana may be encoded by a small gene family. Northern analysis was also carried out to investigate whether there was spatial variation in MS expression among banana organs. It was found that the MaMS probe was hybridized strongly to a single transcript of 1.9 kb in both pulp and peel of the MY fruit, with a higher level of transcripts in the latter (Fig. 4). The size of transcript was comparable to that of MaMS-1, indicating that MaMS-1 is the full-length gene. However, MS transcripts were not detected in other organs such as roots, pseudostem, leaf, male flower and ovary.
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DNA cut with HindIII and GeneRulerTM 1 kb DNA Ladder) are indicated at the left.
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Since MS expressed specifically in fruit tissues, further studies were conducted to investigate whether MS expression was affected temporally during fruit development and ripening. The Northern analysis did not detect MS transcripts in fruit tissues at all developmental stages, ranging from 20–120 DAS (results not shown), indicating that MS does not play a role in fruit development. In fruit harvested at the mature green stage with ripening taking place in the open air at room temperature, it was observed that a total of 23 d was required for ripening to reach the most advanced YB stage. The duration, in terms of DAH required for each ripening stage, was 0 (FG), 9 (TY), 19 (MG), 20 (MY), 21 (GT), 22 (FY), and 23 (YB). However, unlike fruit development, MS transcript could be detected in fruit during ripening, but the level of accumulation was affected by stage of ripening. In general, transcripts in both peel and pulp at the early stages of ripening (FG and TG) were barely detectable, but it began to accumulate in peel as ripening progressed to the MG stage and reached a maximum at MY and other advanced ripening stages (Fig. 5). The pattern of transcript accumulation in pulp differed. The level of transcripts at the MG stage remained low, but it surged to a maximum in MY fruit and declined sharply in fruit at more advanced ripening stages (Fig. 5). In general, transcript accumulation occurred earlier and was more abundant in peel than in the pulp.
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The regulatory role of ethylene on ripening of banana and other climacteric fruits has been well documented (Marriott, 1980; Leliévre et al., 1997). To determine whether ethylene plays a regulatory role in MS expression in banana fruit during ripening in this study, FG fruit was treated for 48 h with different concentrations of exogenous ethylene. Northern analysis showed that MS transcript could be detected in peel of the control fruit (0 µl l–1 ethylene), although transcript in pulp was undetectable (Fig. 5). Nevertheless, the presence of 1 and 20 µl l–1 ethylene stimulated transcript accumulation in both fruit tissues (Fig. 6). The stimulatory effect of ethylene could be inhibited by NBD, as evident from reduced transcript accumulation in fruit treated with both ethylene and NBD. The presence of 2 µl l–1 NBD was shown to decrease transcript abundance partially, while transcript accumulation was completely inhibited by a higher concentration of NBD (20 µl l–1) (Fig. 6).
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Discussion |
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Changes in various biochemical and physiological events in banana fruit during ripening appear to be associated with the activation of a number of genes that are isolated from ripening fruit by differential screening of cDNA libraries (Medina-Suárez et al., 1997; Clendennen and May, 1997; Drury et al., 1999). Some of these genes have been further characterized and the expression has been shown to be related to fruit ripening. These include 1-aminocyclopropane-1-carboxylate (ACC) synthase and ACC oxidase (Liu et al., 1999), sucrose phosphate synthase (do Nascimento et al., 1997), UDPglucose pyrophosphorylase (Pua et al., 2000), pectate lyase (Domínguez-Puigjaner et al., 1997; Pua et al., 2001), metallothionine-like proteins (Liu et al., 2002), and cytochrome P450 (Pua and Lee, 2003). However, isolation and characterization of genes encoding MS have not been reported in banana, although MS has been reported to play a role in gluconeogenesis (Surendranathan and Nair, 1976) and malic acid is a predominant organic acid in banana fruit (Satyan and Parwardhan, 1984; John and Marchal, 1995). In this study, the isolation of the full-length MS cDNA, MaMS-1, encoding a putative MS of 556 amino acid residues from the ripening fruit of banana is reported. MaMS-1 is derived from the mapping of the two partial MS cDNAs, MaMS and a PCR-amplified DNA fragment.
Sequence analysis shows that MaMS-1 is highly homologous (79–81%) to several plant MS homologues. The cDNA also possesses the putative catalytic domain and glyoxysomal targeting signal at the carboxyl terminus, which are conserved in all MS. These sequence characteristics suggest that MaMS-1 encodes a MS in banana. Southern analysis reveals that the MaMS probe strongly hybridizes to 2–4 fragments of genomic DNA digested with various endonucleases that do not cut within the cDNA. This result indicates that MS in banana may be encoded by a small gene family, although the possibility that some DNA fragments might have been generated by the presence of restriction sites in the introns is not ruled out.
The temporal and spatial expression has been carried out to elucidate the role of MS in fruit during development and ripening and in other parts of the plant. Results show no detectable MS transcripts in the fruit at all developmental stages, ranging from 20–120 DAS, suggesting that MS may not play a major role during fruit development of banana. In addition, transcripts cannot be detected in other banana organs (root, pseudostem, leaves, male flower, and ovary) except pulp and peel tissues from the ripening fruit, indicating that MS expression in banana is spatially regulated. Evidence from previous studies indicates that MS expression in organs of light-grown plants is generally low, but is inducible in response to prolonged exposure to darkness (Nieri et al., 1997), starvation (Dieuaide et al., 1992; Graham et al., 1992, 1994) and tissue senescence (De Bellis and Nishimura, 1991; Pistelli et al., 1991, 1996). It is therefore speculated that low MS transcripts in leaves in this study may be attributed to the lack of inducible conditions, for example, starvation and dark treatments. Likewise, the presence of inducible factor(s) may also be responsible for increased MS expression in ripening fruit tissues.
To address the question as to whether MS expression in fruit is associated with ripening, MS is temporally expressed in fruit tissues at different ripening stages. Results show that expression varies with type of fruit tissue and stage of ripening. In general, transcript accumulation occurs earlier and the level is also higher in peel compared to those in pulp, although no transcripts can be detected in both tissues at the early stages of ripening. The onset of transcript accumulation in peel of MG fruit coincided with the climacteric rise of ethylene, as reported previously in this laboratory (Liu et al., 2002). The level of ethylene was generally low in preclimacteric FG and TY fruits, but it increased climactically in MG fruit and declined sharply as ripening progressed to MY and other advanced stages. The rate of ripening appears to be associated with the occurrence of climacteric ethylene. This is evident from the fruit that has taken 19 d to reach the MG stage and only an additional 4 d to be fully ripened. These observations are in line with the previous findings showing that climacteric ethylene plays a pivotal regulatory role in the ripening of banana and other climacteric fruits (Yang and Hoffman, 1984; Leliévre et al., 1997).
Whether ethylene plays a role in MS expression during ripening of banana, transcript abundance in FG fruit was investigated in response to exogenous ethylene alone or in conjunction with NBD. The MS transcript can be detected in control peel but not pulp after 48 h, although no transcript is detected at 0 h in the same organ. There is speculation that low levels of ethylene might have been accumulated in the sealed desiccator that, in turn, up-regulates transcript accumulation in the fruit. This speculation is line with the stimulatory effect of exogenous ethylene at a low level (1 µl l–1) on MS expression in both fruit tissues. The stimulatory effect of ethylene can be suppressed in the presence of NBD, which is the potent inhibitor of ethylene action (Sisler and Yang, 1984), indicating that ethylene plays a regulatory role in MS expression. Furthermore, it may also explain why the accumulation of MS transcript in peel coincides with the climacteric rise of ethylene in MG fruit. However, the mechanism of how ethylene regulates MS expression is not clear. The findings of this study have also prompted speculation that up-regulation of MS transcripts and enzyme activity in senescing tissues (De Bellis and Nishimura, 1991; Pistelli et al., 1991, 1996) or tissues under stress such as dark treatment (Comai et al., 1992; Nieri et al., 1997) and starvation (Dieuaide et al., 1992; Graham et al., 1994) in other studies may be mediated through ethylene, since these tissues have been shown to produce high levels of ethylene (Wright, 1981; Yang and Hoffman, 1984). However, further study is needed to clarify this point.
Increased expression of MS in banana at advanced ripening stages appears to be in line with the accumulation of malate (Satyan and Parwardhan, 1984), which is thought to contribute, in part, to the unique aroma and flavour of the fruit (Grierson and Kader, 1986). Evidence from several lines of study indicates that the up-regulation of MS and other glyoxylate cycle enzymes also plays an important role in gluconeogenesis by the conversion of lipids to carbohydrates and serves as an anaplerotic pathway to supplement intermediates that are usually provided by the TCA cycle (Escher and Widmer, 1997; Eastmond and Graham, 2001). However, the anaplerotic role of the glyoxylate cycle in plants has been disputed, as the TCA cycle may not be needed for the synthesis of carbon skeleton in plant tissues, especially those rich in carbohydrates (Smith, 2002). Increased gluconeogenesis has been observed in banana fruit, where succinate dehydrogenase was inhibited by gamma irradiation but the TCA cycle was not affected (Surendranathan and Nair, 1976). The irradiated fruit also showed an increase in MS and ICL activities and glyoxylate concomitant with a decrease in 
-ketoglutarate and oxaloacetate. While it is not clear whether the lipid content in irradiated fruit is also affected, results of other studies indicate that the level of fruit lipid is relatively constant during ripening (Wade et al., 1980; John and Marchal, 1995). Further work is required to elucidate the precise role of the glyoxylate pathway in banana fruit during ripening.
This study describes, for the first time, the full-length MS cDNA in banana fruit. MS expression in banana is fruit-specific and is regulated by ripening, but not development of the fruit. Unequivocal evidence has also been provided showing that MS expression in fruit during ripening is ethylene-inducible, which may be responsible for the increased accumulation of MS transcripts in climacteric and post-climacteric fruits. These results indicate that malate synthesis in banana fruit during ripening may be regulated, at least in part, by ethylene at the transcriptional level.
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Acknowledgements |
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The authors wish to thank Serena Lim Tze Soo for her excellent technical assistance. This work was supported by research grants, R-154-000-055-112 and R-154-000-077-112, awarded to ECP of the National University of Singapore.
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