Identification of a cis-regulatory element that acts in companion cell-specific expression of AtMT2B promoter through the use of Brassica vasculature and gene-gun-mediated transient assay.

The molecular basis underlying the development, maintenance and function of companion cells in plants is largely unknown. The identification of several genes expressed specifically in companion cells implies the contribution of specific transcriptional elements to the identity of companion cells. However, less is known about the companion cell-specific transcriptional regulation of promoters. We established a novel assay method using gene-gun delivery of partially deleted promoters to string-containing vascular bundles excised from the petiole of Brassica napus for the rapid identification of cis-elements. To test this system, we analyzed the Arabidopsis METALLOTHIONEIN 2B (MT2B) gene, which is expressed in companion cells. The assay revealed a 49-bp region possessing two predicted cis-regulatory elements: a G-box and an evening element-related sequence (EEr), and EEr showed higher activity. We confirmed the reliability of the result with stable transformants harboring a deleted MT2B promoter:GUS transgene. The lack of EEr completely eliminated the MT2B-like expression, but the lack of G-box did not eliminate it. We conclude that EEr is a major cis-regulatory element of the MT2B promoter. Our method will help to explain the transcriptional background of companion cells through the rapid identification of cis-regulatory regions.