The nuclear factor SP8BF binds to the 5′-upstream regions of three different genes coding for major proteins of sweet potato tuberous roots

Abstract

Sporamin and β-amylase are two major proteins of tuberous roots of sweet potato, and expression of genes coding for sporamin and β-amylase is induced concomitantly in leaves with the petioles attached by exogenous supply of sucrose or polygalacturonic acid. We have used a DNase I footprinting assay to characterize nuclear factors that bind to the 5′-upstream regions of gSPO-A1, gSPO-B1 and gβ-Amy genes that encode A-type sporamin, B-type sporamin and the subunit of β-amylase, respectively. Nuclear extracts from sucrose-treated petioles protected a region around -155 relative to the transcription start site of gSPO-A1 and a region around -880 of gβ-Amy from DNase I digestion on both strands. These two protected regions both contained the sequence ACTGTGTA, designated SP8a, in opposite orientation with respect to the direction of transcription. A gel mobility shift assay with SP8a oligonucleotide and competition experiments indicated that a common factor SP8BF binds to the SP8a sequence in gSPO-A1 and gβ-Amy. Binding of SP8BF to the SP8a oligonucleotide was abolished by mutation within the SP8a sequence. Fragments of the 5′-upstream region of gSPO-B1 also competed for the binding of SP8BF to the SP8a oligonucleotide, and the DNase I footprinting assay revealed three binding sites for SP8BF in the 5′-upstream region of gSPO-B1. These three sites in gSPO-B1 all contained the sequence TACTATT, designated SP8b, which shared 4 nucleotides at identical positions with the SP8a sequence. An inverted repeat of the SP8b sequence was also present at one protected site in the 5′-upstream region of gβ-Amy. In addition to sucrose-treated petioles, SP8BF activity was also present in tuberous roots and untreated fresh petioles of sweet potato. Furthermore, the activity was also detected in stems of tobacco plantlets, suggesting that SP8BF is an ubiquitous factor.