Promoter analysis of a type 3 metallothionein-like gene abundant in Satsuma mandarin (Citrus unshiu Marc.) fruit

The genomic region of a type 3 metallothionein (MT3)-like gene (CitMT45) with extensive expression in citrus fruit was isolated from Citrus unshiu Marc. and its promoter activity was analyzed using particle bombardment. A genomic library was screened with CitMT45 cDNA as a probe, and approximately 9.5 kb genomic fragments were obtained. The nucleotide sequence showed that the genomic CitMT45 consists of 3 exons and 2 introns, similar to other plant MT-like genes. Interestingly, the proximal 5′ flanking region had no canonical TATA-box or several cis-regulatory elements of G-box, MRE and ERE motifs common to plant MT-like genes. Lack of the MRE motif in the promoter region is in agreement with no response of CitMT45 to metal treatment, suggesting that CitMT45 might play other biological roles during fruit development rather than detoxification.Two promoters of proximal 190 bp region (PMT45C) and 4.2 kb full region (PMT45L) were fused to the uidA gene and directly incorporated into Citrus tissues using particle bombardment. The PMT45C promoter is sufficient to direct GUS expression in all tested organs and the upstream region from this proximal 190 bp sequence should have cis-elements that act as enhancers in citrus juice sacs. Moreover, a fluorometric GUS analysis of transgenic Arabidopsis indicated that the PMT45L promoter conferred quantitatively preferential expression in siliques. These results might indicate that the cis-elements required for preferential expression in citrus fruits are functionally conserved in heterologous Arabidopsis plants. Thus, it was demonstrated that the PMT45L promoter could be used for manipulation of fruit quality by genetic engineering in Citrus and Arabidopsis.