Journal of Experimental Botany, Vol. 52, No. 362, pp. 1941-1945,
September 1, 2001
© 2001 Oxford University Press
Short Communication |
A fruit-specific and developmentally regulated endopolygalacturonase gene from strawberry (Fragaria x ananassa cv. Chandler)
Departamento de Bioquímica y Biología Molecular, Edificio C-6, Campus Universitario de Rabanales, Universidad de Córdoba, 14071 Córdoba, Spain
Received 27 February 2001; Accepted 8 June 2001
Abstract
A fruit-specific and developmentally regulated polygalacturonase gene (spG gene) from strawberry (Fragariaxananassa cv. Chandler) has been cloned and characterized at a molecular and physiological level. Comparison analysis of the corresponding deduced sPG protein have shown that this strawberry gene is similar to Clade A endopolygalacturonase genes. Moreover, the spatio-temporal and hormonal gene expression pattern suggests a close relationship between the expression of this gene and the onset of the strawberry fruit ripening process and agrees with that of the production of oligosaccharins which have already been described as active molecules involved in fruit ripening. The results are discussed in terms of a putative role of this enzyme in the release of oligosaccharins from the strawberry fruit cell wall.
Key words: Strawberry, fruit-ripening, endopolygalacturonase.
Introduction
In fruits, late softening and tissue deterioration are associated both with extensive pectin cell wall disassembly in the later stages of ripening and with fruit degradation in the overripe stages (Hadfield and Bennett, 1998
). A wide range of cell wall hydrolase enzymes (xylanases, exo- and endopolygalacturonases, ß-galactanases, ß-galactosidases, mannanases, cellulases, pectin methylesterases, and pectate lyases) are known to catalyse different aspects of pectin modification and disassembly of the cell wall (Fry, 1995; Medina-Escobar et al., 1997; Brownleader et al., 1999). Of these enzymes, the best characterized are exo- and endopolygalacturonases (PGs). These have been related, in tomato and other fruits, with the reduction of the apparent molecular size of pectic polymers by cleaving the neutral side-chain residues of the cell wall pectins as fruit ripens (Hadfield and Bennett, 1998, and references therein). Recently, a genetic linkage between freestone character and endo-PG activity has been identified in peach (Lester et al., 1994). Also, a correlation between pectin solubilization and depolymerization of pectins and an increase in pectin-degrading enzyme activity and in the appearance of PG mRNAs has been shown (Hadfield and Bennett, 1998). Moreover, using antisense tomato plants with decreased levels of PG mRNA, or the pleiotropic tomato mutant ripening inhibitor (rin), it has been shown that PG-mediated pectin depolymerization is not necessary for normal fruit ripening and softening, and so indicating that other cell wall enzymes are also involved in these processes (Hadfield and Bennett, 1998, and references therein). However, the results of these experiments are consistent with the suggestion that the PG-mediated pectin disassembly does not contribute to early fruit softening, but contributes significantly to tissue deterioration in the late stages of fruit ripening. Nevertheless, inhibition of PG leads to modest improvements in shelf-life and the consistency of processed tomato juice (Hadfield and Bennett, 1998). Therefore, these results suggest both that PG-mediated pectin disassembly is important for tissue alterations only in the last stages of maturation and that polygalacturonase is not the only enzyme activity responsible for pectin solubilization, but it is responsible for the reduction in molecular weight of polyuronides during ripening (Hadfield and Bennett, 1998). Additionally, it has also been suggested that polygalacturonase action on the pectin network may enhance the action of other cell wall-degrading enzymes by increasing the accessibility of such enzymes to their substrates (Redgwell et al., 1997).
In strawberry, three different polygalacturonase activities have been partially characterized so far (Nogata et al., 1993). Two of them, PG2 and PG3, have been shown to have exopolygalacturonase activity, whereas PG1 presented both a strong exopolygalacturonase activity and a weak endopolygalacturonase activity (Nogata et al., 1993). However, the three isoenzymes were purified from small green stage fruit (Nogata et al., 1993). Moreover, the total exopolygalacturonase activity decreased during fruit growth and ripening (Nogata et al., 1993).
In the present paper, the cloning, molecular and physiological characterization of a strawberry endopolygalacturonase gene (spG gene), whose expression pattern strongly suggests a relationship of this gene with the fruit ripening process, is reported. Furthermore, the putative involvement of this gene in the releasing of oligosaccharins as biologically active molecules involved in fruit ripening is also discussed.
Materials and methods
Plant material and hormonal treatments
The strawberry fruit (Fragariaxananassa cv. Chandler) was harvested at different developmental stages as previously described (Medina-Escobar et al., 1997): small-sized green fruits (G1), middle-sized green fruits (G2), full-sized green fruits (G3), white fruits with green achenes (W1), white fruits with red achenes (W2), turning stage fruits (T), and full-ripe red fruits (R).
For the hormonal treatments, achenes from two sets of G2-stage strawberry fruits were carefully removed from the growing plant using the tip of a scalpel blade as previously published (Medina-Escobar et al., 1997). Briefly, one set of de-achened fruits was treated with the synthetic auxin 1-naphthaleneacetic acid (NAA) as a lanolin paste with 1 mM NAA in 1% (v/v) DMSO. The other set of de-achened fruits (control group) were treated with the same paste but without NAA. Both treatments were applied over the whole fruit surface. Fruits were kept on the plant in the experimental field and harvested 0, 24, 48, 72, and 96 h after treatment and immediately frozen in liquid nitrogen and stored at -80 °C.
Isolation of the strawberry spG-cDNA
The spG-cDNA was cloned using the differential display PCR technique (DD/RT-PCR) and two different strawberry fruit RNA populations were compared: one isolated from green fruits and the other from de-achened green fruits. The screening for true positives was performed by reverse Northern blots. The positive cDNA fragments were then cloned into pGEM-T Easy vectors (Promega) according to the manufacturer's protocol, and sequenced to completion.
RNA isolation and Northern analysis
Total RNA from fruits at the stages indicated above, and from roots, leaves, flowers, and runners was isolated according to Manning (Manning, 1991). Northern analysis and auxin treatments were as described by Medina-Escobar et al. (Medina-Escobar et al., 1997). The blot was hybridized using the complete 588 bp spG-cDNA fragment described in Fig. 1 as a radioactive probe. A cDNA corresponding to 18S rRNA was used as a ribosomal probe to control equal loading of RNA samples. The probes were labelled to a specific activity of c. 108 cpm µg-1 using a commercial random priming kit (Amersham-Pharmacia).
|
Cloning of the genomic spG gene
A 
-FIX genomic library from Fragariaxananassa cv. Chandler was screened following standard procedures. The 32P-labelled 588 bp spG-cDNA fragment (Fig. 1
) was used as a probe to screen approximately 400 000 recombinant phages.
DNA isolation and Southern analysis
Strawberry genomic DNA was extracted from achenes removed from W1-stage strawberry fruits as described previously (Medina-Escobar et al., 1997). DNA (5 µg) was digested with different restriction enzymes and Southern analysis was performed (Medina-Escobar et al., 1997).
Results and discussion
By means of the DD/RT-PCR technique and comparing mRNA populations isolated from green stage strawberry fruit (G2) and from strawberry G2-de-achened fruit, a partial cDNA fragment corresponding to a strawberry mRNA encoding an endopolygalacturonase (spG-cDNA) was isolated. The screening of a strawberry genomic library with the spG-cDNA fragment as a probe allowed a genomic clone (spG gene) carrying the complete structural strawberry gene and about 850 bp of its 5'-flanking promoter region to be isolated (Fig. 1). Both the isolated spG-cDNA fragment and the spG gene were shown to be 100% identical at the nucleotide level. An ORF encoding a 401 amino acid protein was deduced from the spG gene sequence (Fig. 1). Comparison of the sPG deduced protein with those present in the databases revealed a significant amino acid sequence identity of this strawberry protein with sequences corresponding to higher plant endopolygalacturonases (Fig. 2). As in all higher plant endopolygalacturonases, the strawberry protein sPG contains an N-terminal hydrophobic signal peptide with a potential cleavage site between amino acid residues 22 and 23. The cleavage of the signal peptide results in a mature protein of 40.69 kDa, with a pI of 7.91. Potential N-glycosylation sites (N-X-S/T) are also found at amino acid positions 212 and 333. According to the amino acid sequence and the level of similarity and identity found with other PGs (Fig. 2), the sPG protein can be classified within the Clade A PG polygalacturonases which includes PG proteins related to fruit ripening and abscission processes (Hadfield and Bennett, 1998). Thus, the four conserved domains found in all eukaryotic and prokaryotic polygalacturonase sequences and proposed to be involved in PG activity are also present within the sPG protein (domains I to IV). Furthermore, a highly conserved His residue (GHG motifs, in domain III; residue 245), assigned to a catalytic function in all PGs sequenced to date, and a Tyr residue (amino acid position 315) which is strictly conserved in PGs and has also been shown to be essential for the activity of PG (Hadfield and Bennett, 1998), is also present at the same position in the strawberry endopolygalacturonase (Fig. 2). In addition, the eight cysteine residues conserved between the PGs from higher-plant species and fungi, are also observed in the strawberry sPG protein (Fig. 2).
|
The hybridization pattern observed for the spG gene in Southern blot experiments, suggests the presence of a small PG multigene family in the strawberry genome (data not shown). Alternatively, this pattern could also be due to the ploidy level of the strawberry.
The spatio-temporal and hormonal expression pattern of the spG gene has been studied. As shown in Fig. 3A, a fruit-specific expression pattern was observed for this strawberry gene. Moreover, the spG gene was exclusively expressed in two different fruit-ripening stages (W1 and W2), showing the highest transcript level in the W2 stage (Fig. 3A). However, no spG gene expression during the fruit elongation stages G1, G2, and G3 or during the late ripening stages, T and R, was detected. Though the presence of three PGs isoenzymes was described in strawberry fruits, these enzymes were purified from small green-stage fruit (Nogata et al., 1993). At this strawberry fruit stage no mRNA expression corresponding to the spG gene described here was detected, thus strongly supporting the fact that the weak endopolygalacturonase activity described for the PG1 isoenzyme by Nogata et al. (Nogata et al., 1993) does not correspond to the protein encoded for the strawberry sPG gene described in this article. Such a gene expression pattern suggests a close relationship between the physiological role of the sPG protein and the onset of the fruit ripening process. In fact, during fruit ripening, it is clearly established that cell wall degradation leads to fruit softening (Perkins Veazie, 1995). At present, and similarly to other fruits, some cell wall-hydrolytic enzymes such as pectin methyl esterase, pectate lyase and cellulase have been implicated in strawberry fruit softening (Perkins Veazie, 1995; Medina-Escobar et al., 1997; Trainotti et al., 1999). Besides, a strong induction in the expression of genes encoding cell wall-degrading enzymes such as cellulase and pectate lyase has also been observed during all the stages of strawberry fruit ripening and softening: stages W2, T, and R (Medina-Escobar et al., 1997; Trainotti et al., 1999). However, the expression pattern found for the spG gene is quite intriguing and does not support a clear relationship between its expression and the process of fruit softening. Accordingly, only a high expression transcript level was detected in the first stage of fruit ripening (W2 stage) where no strong and evident softening is yet produced in the strawberry fruit. Furthermore, healthy fruits, abscission zones, and fungal and bacterial phytopathogens, all produce endo-PG activities. These enzymes act on pectins in vivo to generate biologically active oligosaccharins (oligo-GalAs) (Dumville and Fry, 2000). Also, in healthy ripening fruits there is strong evidence supporting the production of oligogalacturonides in vivo which have been involved in a signalling role in non-diseased plant tissues (Dumville and Fry, 2000). Thus, the application of oligo-GalAs to growing tissues has an effect on diverse processes such as cell expansion, transport, morphogenesis, and fruit ripening (Dumville and Fry, 2000). Also, it has been shown that the external application of pectic oligosaccharins induces fruit ripening in tomato and citrus fruits (Dumville and Fry, 2000). Therefore, the spatio-temporal expression pattern found for the strawberry spG gene is quite consistent with a putative role of the corresponding sPG endopolygalacturonase in the production of oligalacturonides oligosaccharins. Thus, the suddenly and narrow gene expression peak at the very beginning of the fruit-ripening process (W1 and W2 stages) supports this hypothesis. Consequently, these signalling molecules could be potentially involved in the regulation of some metabolic processes related to strawberry fruit ripening as already proposed for tomato fruit (Dumville and Fry, 2000). Alternatively, the sPG protein might be involved in the hydrolysis of some very specific bonds within the cell wall pectin network, which can be the first step and necessary for subsequent and generalized cell wall pectins degradation by other cell wall-degrading enzymes such as pectate lyases (Medina-Escobar et al., 1997; Trainotti et al., 1999).
|
It has previously been shown that, in strawberry fruit, the expression of some ripening-related genes are directly or indirectly negatively regulated by the auxins produced in the achenes. Auxins stimulate expansion of the receptacle, but inhibit ripening (Perkins Veazie, 1995
; Medina-Escobar et al., 1997). Thus, in order to ascertain if the strawberry spG gene was also under hormonal control produced by the achenes, Northern experiments were performed with RNA isolated from achened fruit and de-achened fruit treated with or without NAA. Molecular studies have shown that in strawberry the ripening process began with the B2 stage of fruit development (Medina-Escobar et al., 1997). Also, the removal of the achenes in the G2 stage of fruit growth induces the expression of genes specifically involved in the ripening process (Medina-Escobar et al., 1997). A green stage G2 was selected for these studies as achenes are not yet matures and are actively producing auxins which later drop rapidly both in the receptacle and in the achenes, after the white stage (W1-W2 stages) (Perkins Veazie, 1995). Curiously, at these white stages (W1, W2) the expression of the spG gene peaks (Fig. 3A) so suggesting a regulation of this gene by the achenes. Therefore, the effect of removing achenes from the fruit on this spG gene expression can be well distinguished at the green stage (G2), as has already been done for other strawberry genes (Medina-Escobar et al., 1997), as no expression of this gene seems yet to have taken place (Fig. 3A, lane 3). As shown in Fig. 3B, a clear increase in spG gene transcript level was found in strawberry G2 stage de-achened fruits after 5 d. Furthermore, this increase in spG transcript levels was nearly completely reverted in G2 de-achened fruit treated with NAA after 5 d, and so strongly suggesting that the expression of the spG gene is under the control of auxins. Similar results have been shown for other strawberry ripening-related genes involved in cell wall disassembly, such as pectate lyase and cellulase (Medina-Escobar et al., 1997; Trainotti et al., 1998), as well as for those related to other ripening-related metabolic processes (Manning, 1998). In this sense, several lines of evidence suggest that pectic oligosaccharins antagonize the action of auxins on physiological processes such as auxin-induced growth of pea stem segments, auxin-stimulated rooting in leaf explants, and auxin-dependent somatic embryogenesis when applied at very low level (Creelman and Mullet, 1997). Similar antagonistic results were found with a PG gene involved in the abscission process where an IAA treatment inhibited the gene expression in the abscission zone (Kalaitzis et al., 1995). In order to clarify the role of the strawberry spG gene in the strawberry fruit ripening process, experiments with antisense strawberry transgenic plants are underway.
Acknowledgments
This work was supported by grant CICYT BIO98-0496-C02-02 (Spain) and Junta de Andalucía (Grupo CVI 115), Spain. The utilization of equipment of Instituto Andaluz de Biotecnología, Andalucía, Spain, is also acknowledged. EM thanks the Ministerio de Educacion y Ciencia (Spain) for a post-doctoral contract and J Redondo-Nevado also thanks New Biotechnic for a post-doctoral fellowship. The spG gene sequence is under patent P200000579.
Notes
1 To whom correspondence should be addressed. Fax: +34 957218 592. bb1mublj{at}uco.es 
References
Brownleader MD, Jackson P, Mobasheri A, Pantelides AT, Sumar S, Trevan M, Dey PM. 1999. Molecular aspects of cell wall modifications during fruit ripening. Critical Review of Food Science Nutrition 39, 149–164.
Creelman RA, Mullet JE. 1997. Oligosaccharins, brassinolides, and jasmonates: non-traditional regulators of plant growth, development and gene expression. The Plant Cell 9, 1211–1233.[Web of Science][Medline]
Dumville JC, Fry SC. 2000. Uronic acid-containing oligosaccharins: their biosynthesis, degradation and signalling role in non-diseased plant tissues. Plant Physiology and Biochemistry 38, 125–140.
Fry SC. 1995. Polysaccharide-modifying enzymes in the plant cell wall. Annual Review of Plant Physiology and Plant Molecular Biology 46, 497–520.[Web of Science]
Hadfield KA, Bennett AB. 1998. Polygalacturonases: many genes in search of a function. Plant Physiology 117, 337–343.
Kalaitzis P, Koeheler SM, Tucker ML. 1995. Cloning of a tomato polygalacturonase expressed in abscission. Plant Molecular Biology 28, 647–656.[Web of Science][Medline]
Lester DR, Speirs J, Orr G, Brady CJ. 1994. Peach (Prunus persica) endopolygalacturonase cDNA isolation and mRNA analysis in melting and non-melting peach cultivars. Plant Physiology 105, 225–231.[Abstract]
Manning K. 1998. Isolation of a set of ripening-related genes from strawberry: their identification and possible relationship to fruit quality traits. Planta 205, 622–631.[Web of Science][Medline]
Medina-Escobar N, Cárdenas J, Moyano E, Caballero JL, Muñoz Blanco J. 1997. Cloning, molecular characterization and expression pattern of a strawberry ripening specific cDNA with sequence homology to pectate lyase from higher plants. Plant Molecular Biology 34, 867–877.[Web of Science][Medline]
Nogata Y, Ohta H, Voragen AGJ. 1993. Polygalacturonase in strawberry fruit. Phytochemistry 34, 617–620.
Perkins Veazie P. 1995. Growth and ripening of strawberry fruit. Horticultural Reviews 17, 267–197.
Redgwell RJ, MacRae E, Hallet I, Fischer M, Perry J, Harker R. 1997. In vivo and in vitro swelling of cell walls during fruit ripening. Planta 203, 162–173.
Trainotti L, Spolaore S, Pavanello A, Baldan B, Casadoro G. 1999. A novel E type endo-ß-1,4-glucanase with a putative cellulose-binding domain is highly expressed in ripening strawberry fruits. Plant Molecular Biology 40, 323–332.[Web of Science][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  M. A. Quesada, R. Blanco-Portales, S. Pose, J. A. Garcia-Gago, S. Jimenez-Bermudez, A. Munoz-Serrano, J. L. Caballero, F. Pliego-Alfaro, J. A. Mercado, and J. Munoz-Blanco Antisense Down-Regulation of the FaPG1 Gene Reveals an Unexpected Central Role for Polygalacturonase in Strawberry Fruit Softening Plant Physiology, June 1, 2009; 150(2): 1022 - 1032. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Castillejo, J. I. de la Fuente, P. Iannetta, M. A. Botella, and V. Valpuesta Pectin esterase gene family in strawberry fruit: study of FaPE1, a ripening-specific isoform J. Exp. Bot., April 1, 2004; 55(398): 909 - 918. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Benitez-Burraco, R. Blanco-Portales, J. Redondo-Nevado, M. L. Bellido, E. Moyano, J.-L Caballero, and J. Munoz-Blanco Cloning and characterization of two ripening-related strawberry (Fragariaxananassa cv. Chandler) pectate lyase genes J. Exp. Bot., February 1, 2003; 54(383): 633 - 645. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Aharoni and A. P. O'Connell Gene expression analysis of strawberry achene and receptacle maturation using DNA microarrays J. Exp. Bot., October 1, 2002; 53(377): 2073 - 2087. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||