Expression Analysis of a Ripening-Specific, Auxin-Repressed Endo-1,4-β-Glucanase Gene in Strawberry
Abstract
A cDNA (Cel1) encoding an endo-1,4-β-glucanase (EGase) was isolated from ripe fruit of strawberry (Fragaria × ananassa). The deduced protein of 496 amino acids contains a presumptive signal sequence, a common feature of cell wall-localized EGases, and one potential N-glycosylation site. Southern- blot analysis of genomic DNA from F. × ananassa, an octoploid species, and that from the diploid species Fragaria vesca indicated that the Cel1 gene is a member of a divergent multigene family. In fruit, Cel1 mRNA was first detected at the white stage of development, and at the onset of ripening, coincident with anthocyanin accumulation, Cel1 mRNA abundance increased dramatically and remained high throughout ripening and subsequent fruit deterioration. In all other tissues examined, Cel1 expression was invariably absent. Antibodies raised to Cel1 protein detected a protein of 62 kD only in ripening fruit. Upon deachenation of young white fruit to remove the source of endogenous auxins, ripening, as visualized by anthocyanin accumulation, and Cel1 mRNA accumulation were both accelerated. Conversely, auxin treatment of white fruit repressed accumulation of both Cel1 mRNA and ripening. These results indicate that strawberry Cel1 is a ripening-specific and auxin-repressed EGase, which is regulated during ripening by a decline in auxin levels originating from the achenes.
Fruit ripening is a complex developmental program in which senescing tissues undergo programmed changes in firmness, texture, coloration, flavor, and susceptibility to microbial infection. Changes in firmness and texture are largely attributed to alterations in the composition and structure of cell wall polysaccharides. Because these modifications influence the postharvest properties (i.e. storage time and expense, handling damage, and desirability to the consumer) of important food crops and, consequently, are of great commercial importance, research in recent years has focused on identifying enzyme activities that are rate limiting in the promotion of fruit deterioration. In the climacteric species, which are characterized by the autocatalytic production of the ripening hormone ethylene and a ripening-related transient burst in CO2 evolution, the antisense suppression of ACC synthase (Oeller et al., 1991) and ACC oxidase (Picton et al., 1993) in tomato has provided fruit in which ripening and softening are retarded and can be controlled by the application of ethylene. Similar approaches have been taken in efforts to diminish the activities of cell wall-associated hydrolases (Sheehy et al., 1988; Smith et al., 1988), which may play a central role in fruit cell wall breakdown during ripening (Brady, 1987).
In nonclimacteric species such as strawberry (Fragaria × ananassa), much less is known about the ripening process. Because these plants lack a respiratory climacteric and because ethylene appears to play a minimal, if any, role in fruit ripening, there is growing interest in identifying the factor(s) that mediates ripening. Strawberry fruit (actually an enlarged receptacle rather than a true fruit) exhibit a low level of ethylene production, which is rather constant during ripening (Knee et al., 1977), and there is no observable stimulation of ripening upon application of exogenous ethylene (Iwata et al., 1969). Although there is no evidence for a ripening-related role for ethylene, strawberry fruit ripening has been shown to be negatively regulated by auxins that originate in the receptacle-borne achenes (Given et al., 1988b; Manning, 1994). As auxin levels decline, fruit exhibit a characteristic ripening profile, one of the major hallmarks of which is rapid deterioration once fruit achieve the red-ripe stage. In general, strawberry fruit ripening is typified by the induction of enzyme markers for anthocyanin pigment biosynthesis (e.g. Phe ammonia-lyase), a concomitant decrease in chlorophyll and increase in anthocyanin pigments, and a progressive decrease in tissue firmness (Woodward, 1972; Given et al., 1988a).
Efforts to reveal the molecular basis of changes in firmness, which are a major contributing factor to fruit quality, have focused on cell wall-associated enzymes, which are believed to mediate and/or contribute to cell wall breakdown. The most studied of these activities, endopolygalacturonase, is absent or below the limit of detection in ripening strawberry fruit (Neal, 1965; Barnes and Patchett, 1976; Huber, 1984). Although strawberry fruit is a rich source of pectin, this observation is consistent with cell wall studies that have shown that total extractable polyuronides remain constant as a proportion of cell wall material during ripening and do not show detectable depolymerization (Huber, 1984). In contrast to these findings, however, the hemicellulosic fraction of cell walls prepared from ripening fruit demonstrates a progressive shift from high- to low-Mr polymers (Huber, 1984). Whereas there is no discernible change in the neutral sugar composition of hemicelluloses isolated from the small-sized green to red-ripe stages, the average net Mr change is quite dramatic, suggestive of an active, developmentally regulated endohydrolyase. It is interesting that this observed hemicellulose depolymerization correlates well with a soluble CMCase activity measured in extracts prepared from ripening strawberry fruit (Barnes and Patchett, 1976). In ripening fruit of avocado (Hatfield and Nevins, 1986) and pepper (Harpster et al., 1997), CMCase activity is attributed to an EGase (EC 3.2.1.4).
Although largely correlative, there is considerable evidence for the importance of EGases in a wide variety of physiological processes involving changes in cell wall architecture, which range from cell wall expansion to disassembly (for review, see Brummell et al., 1994). For example, in abscission-zone formation the infusion of antiserum raised against an abscission-zone-related EGase into explants, which had been induced to abscise by ethylene, was observed to inhibit cell separation (Sexton et al., 1980). Furthermore, the induction of EGase gene expression in fruit of tomato (Lashbrook et al., 1994), avocado (Christoffersen et al., 1984), and pepper (Ferrarese et al., 1995; Harpster et al., 1997) correlates well with the onset and development of ripening. Recently, a partial cDNA showing homology to EGases was isolated from strawberry fruit and shown to be expressed in a ripening-related manner (Manning, 1998). As a first step toward determining whether the in vivo suppression of EGase gene expression is a viable strategy for enhancing firmness in harvested strawberry fruit, we describe the cloning of a full-length strawberry EGase cDNA (Cel1), an analysis of the hormonal and developmental regulation of Cel1 gene expression, and the identification and quantitation of Cel1 protein.
ACKNOWLEDGMENTS
We thank Dr. Rita Teutonico for her efforts in the construction of the cDNA library, Malini Nag for assistance with the isolation and characterization of cDNA clones, Dr. Paul Oeller for careful review of the manuscript, and Jay Maddox and Vincenzo Pirozzi of the greenhouse staff for maintenance of the plants.
Abbreviations:
| CMCase | carboxymethylcellulase |
| dpa | days postanthesis |
| EGase | endo-1,4-β-glucanase |
| NAA | naphthalene acetic acid |
| ORF | open reading frame |
Footnotes
The accession number for the sequence reported in this article is {"type":"entrez-nucleotide","attrs":{"text":"AF074923","term_id":"3549290"}}AF074923.