Presence and expression of NAD+-dependent sorbitol dehydrogenase and sorbitol-6-phosphate dehydrogenase genes in strawberry

The sorbitol metabolic pathway in strawberry (Fragaria X ananassa Duch. var.'Nyoho') is unclear, despite strawberry belonging to the family Rosaceae. Therefore, the presence or absence of genes for NAD(+)-dependent sorbitol dehydrogenase (NAD-SDH) and sorbitol-6-phosphate dehydrogenase (S6PDH) in strawberry, and their levels of expression related to growth were investigated. A full-length cDNA encoding the NAD-SDH gene in strawberry (FaSDH) was obtained using rapid amplification of cDNA ends (RACE). The full-length clone consisted of 1,399 bp and contained a 1,083 bp open reading frame encoding 361. amino acids with a calculated molecular mass of 38.8 kDa, and a predicted isoelectric point (pI) of 6.08. This nucleotide sequence showed 79 - 85% similarity with the NAD-SDH genes of other plants. Its amino acid sequence contained both zinc- and NAD-binding sites, confirming that this sequence belonged to the NAD-SDH gene family. Southern blotting suggested that the genomic copy number of FaSDH was less than in apple. The expression of FaSDH transcripts in strawberry fruit was higher at the young and mature stages than at other stages. The transcript level in mature leaves was higher than in the shoot tip, and in folded and young leaves. NAD-SDH activity was detected in strawberry fruits and leaves and the activity in fruit was comparable to that in other fruits. Reverse transcriptase (RT)-PCR using specific primers designed based on the S6PDH gene of Fragaria vesca, showed a clear single band in leaves and fruits of strawberry. Sequence analysis showed this RT-PCR fragment, named FaS6PDH, to have high homology to the S6PDH gene sequence of other plants, suggesting that the S6PDH gene exists in the strawberry genome and that its mRNA is expressed in leaves and fruits. However, neither the protein nor its enzyme activity were detected in leaves or fruit.