An expansin gene expressed in ripening strawberry fruit

Abstract

Tissue softening accompanies the ripening of many fruit and initiates the processes of irreversible deterioration. Expansins are plant cell wall proteins proposed to disrupt hydrogen bonds within the cell wall polymer matrix. Expression of specific expansin genes has been observed in tomato (Lycopersicon esculentum) meristems, expanding tissues, and ripening fruit. It has been proposed that a tomato ripening-regulated expansin might contribute to cell wall polymer disassembly and fruit softening by increasing the accessibility of specific cell wall polymers to hydrolase action. To assess whether ripening-regulated expansins are present in all ripening fruit, we examined expansin gene expression in strawberry (Fragaria x ananassa Duch.). Strawberry differs significantly from tomato in that the fruit is derived from receptacle rather than ovary tissue and strawberry is non-climacteric. A full-length cDNA encoding a ripening-regulated expansin, FaExp2, was isolated from strawberry fruit. The deduced amino acid sequence of FaExp2 is most closely related to an expansin expressed in early tomato development and to expansins expressed in apricot fruit rather than the previously identified tomato ripening-regulated expansin, LeExp1. Nearly all previously identified ripening-regulated genes in strawberry are negatively regulated by auxin. Surprisingly, FaExp2 expression was largely unaffected by auxin. Overall, our results suggest that expansins are a common component of ripening and that non-climacteric signals other than auxin may coordinate the onset of ripening in strawberry.

Figure 1

A, Deduced amino acid sequence from FaExp2. The site of cleavage of putative signal sequence is indicated by an arrow. Conserved Cys residues found in all sequenced expansins are marked by an asterisk below, and conserved Trp residues by a cross above the sequence. B, Phylogenetic tree of full-length deduced amino acid sequences of 24 expansins using Lolium perenne pollen allergen (M57474) as an outgroup. Alignments was made using ClustalV multiple sequence software (Higgins et al., 1992) and phylogenetic relationships defined by PAUP software using a heuristic search with 100 replicates. A single tree was obtained (l = 1189, ci = 0.675, ri = 0.587). Bootstrap values are indicated in bold above, and branch lengths are indicated below the branches. Deduced amino acid sequences and GenBank accession numbers are: Arabidopsis, AtExp2 (U30481), Exp5 (U30478), and Exp6 (U30480); Brassica napus, BnExp (AJ000885); Cucumis sativus, CsExp1 (U30382) and CsExp2 (U30460); Fragaria × ananassa, FaExp2 (AF159563); Gossypium hirsutum, GhExp (AF043284); Lycopersicon esculentum, LeExp1 (U82123), LeExp3 (AF059487), LeExp4 (AF059488), LeExp5 (AF059489), and LeExp18 (AJ004997); Nicotiana tabacum, NtExp1 (AF049350), NtExp2 (AF049351), NtExp3 (AF049352), and NtExp4 (AF049353); Oryza sativa, OsExp1 (Y07782), OsExp2 (U30477), OsExp3 (U30479), and OsExp4 (U85246); Prunus armeniaca, PaExp1 (U93167) and PaExp2 (AF038815); and Pisum sativum, PsExp (X85187).

Figure 2

Southern blot of genomic DNA from strawberry. Genomic DNA (20 μg per lane) was digested with the indicated restriction enzymes and hybridized with a ³²P-labeled piece of FaExp2 probe (probe 2). The blot was washed three times for 20 min at 55°C with 0.2× SSC, 0.1% (w/v) SDS, and 0.01% (w/v) sodium PPi, and then exposed to a phosphor-imaging plate and analyzed with a phosphor imager.

Figure 3

RNA gel-blot analysis of FaExp2 mRNA abundance in fruit and vegetative tissues. Total RNA (10 μg) from roots (R), stems (ST), leaves (L), sepals (S), ovaries (O), green achenes (GA), and ripe receptacle (RR) was electrophoresed and then hybridized with probe 2. Blots were washed three times for 20 min at 55°C with 0.2× SSC 0.1% (w/v) SDS, and 0.01% (w/v) sodium PPi. The blot was exposed to x-ray film with an intensifying screen at −80°C.

Figure 4

A, FaExp2 expression throughout strawberry fruit ripening. Northern-blot analysis of total RNA (10 μg) extracted from fruits at the following stages: small green (SG), large green (LG), white (W), turning (T), ripe (R), and overripe (OR). The blot was hybridized with probe 2. B, Western blot of total proteins extracted from ripening fruit were separated by SDS-PAGE and visualized by reaction with antibodies to LeExp1. C, Firmness evolution through strawberry fruit ripening. Fruits were compressed 0.5 mm and the maximum force reached was registered. Data were analyzed by ANOVA and the means compared by lsd_(0.05) test. Letters above the bars indicate statistically significant (P < 0.05) differences between data groups.

Figure 5

Effect of auxins on FaExp2 expression. Northern-blot analysis of total RNA (10 μg) extracted from white (W) or turning (T) fruits after 3 d at 20°C under different auxin conditions. These conditions resulted from combining the presence (+) or absence (−) of achenes with the application (+) or no application (−) of 1 mm NAA. The blot was hybridized with probe 2 for FaExp2. As a control for the auxin treatment, the same blot was stripped and subsequently hybridized with a probe for FaCel1, an auxin-repressed gene (Harpster et al., 1998).

Figure 6

Effect of ethylene on FaExp2 expression. Northern-blot analysis of total RNA (10 μg) extracted from white fruits maintained 3 d at 20°C in a continuous flow (20 L h⁻¹) of humidified air in presence or absence of 10 μL L⁻¹ of ethylene. The same blot was hybridized with probes for FaExp2 (probe 2) and FaCel1 (probe 3).