A fruit-specific putative dihydroflavonol 4-reductase gene is differentially expressed in strawberry during the ripening process

Abstract

A cDNA clone encoding a putative dihydroflavonol 4-reductase gene has been isolated from a strawberry (Fragaria x ananassa cv Chandler) DNA subtractive library. Northern analysis showed that the corresponding gene is predominantly expressed in fruit, where it is first detected during elongation (green stages) and then declines and sharply increases when the initial fruit ripening events occur, at the time of initiation of anthocyanin accumulation. The transcript can be induced in unripe green fruit by removing the achenes, and this induction can be partially inhibited by treatment of de-achened fruit with naphthylacetic acid, indicating that the expression of this gene is under hormonal control. We propose that the putative dihydroflavonol 4-reductase gene in strawberry plays a main role in the biosynthesis of anthocyanin during color development at the late stages of fruit ripening; during the first stages the expression of this gene could be related to the accumulation of condensed tannins.

Figure 1

Nucleotide and deduced amino acid sequence from strawberry cDNA clone njjs24 (accession no. AF029685). Initiation and stop codons are shown in bold. The putative polyadenylation sequence is underlined. Asterisks (*) indicate amino acid residues conserved in the hydroxysteroid dehydrogenase/DFR superfamily (Baker and Blasco, 1992). The box located in the N-terminal region shows the putative NADP-binding domain. A second box shows the stretch thought to define the substrate specificity of the DFR enzyme (Beld et al., 1989).

Figure 2

Comparison of the amino acid sequences of the putative strawberry DFR with that of higher plants. Identical and conservative amino acids found in all DFR sequences are shown in black; those in four or more sequences are shown in gray. The amino acid region determining the substrate specificity is underlined. The accession numbers are X15536 for A. majus (ampallid); X15537 for P. hybrida (phybrida); AF029685 for F. ananassa (dhfrt7); D85102 for R. hybrida (rosa); X75964 for V. vinifera (vvinifera); P51102 for A. thaliana (athaliana); and Z67983 for D. caryophyllus (dcaryophyl). Alignment was performed using the GenDoc program (version 2.0.004, 1997) by K.B. Nicholas and H.B. Nicholas, Jr.

Figure 3

Northern analysis of 20 μg of total RNA isolated from pooled samples of G1, G2, G3, W1, W2, T, and R strawberry fruit stages and from roots (Rt), leaves (L), flowers (F), and stolons (St). The size of hybridizing transcript is indicated in kilobase pairs. A, Hybridization was to a ³²P-labeled njjs24 probe. B, The same blot was hybridized with a ³²P-labeled 18S RNA probe used as a control.

Figure 4

Northern analysis of 20 μg of total RNA isolated from receptacle (de-achened fruit) at the same developmental stages indicated in Figure 3. A and B are as described in Figure 3.

Figure 5

Effects of removing achenes and treatment with auxin on putative strawberry DFR gene expression. Northern analysis of total RNA (20 μg) isolated from G2-stage strawberry fruit at 0 h (lane 1), 24 h (lane 2), 48 h (lane 3), 72 h (lane 4), and 96 h (lane 5) after removing the achenes (Ac). Lane 6, G2-stage de-achened fruit treated with NAA at 96 h. Lane 7, G2-stage strawberry fruit with achenes. A and B are as described in Figure 3.

Figure 6

Southern blot of strawberry genomic DNA digested with several restriction enzymes. Total DNA (4 μg) was extracted from achenes, electrophoresed, blotted, and hybridized with the ³²P-labeled njjs24 probe.