Extensins are hydroxyproline-rich glycoproteins, which are abundant in the cell wall of most plant species. It has been found that expression of extensin genes is induced in areas of the plant that are subjected to mechanical stresses. The promoter of the extA extensin gene from oilseed rape (Brassica napus) has been previously characterised and a negative regulatory region (NRR), which repressed expression in non-phloem cell types, was located between -664 and -433bp relative to the transcription start site. This repression was shown to be overcome in regions of the plant subjected to mechanical stresses, resulting in high expression levels.
In the current work, the NRR from the extA gene promoter was dissected
further using a series of PCR-generated 5' promoter truncations fused to the β-glucuronidase (gus) reporter gene. These truncations terminated at -636bp, -619bp, -586bp, -569bp, -547bp, -524bp, -505bp, -481bp and -452bp, relative to the transcription start site. Transgenic tobacco plants containing these truncations were regenerated and extA-driven gus expression was analysed.
Histochemical analysis of mature stem cross sections from transgenics
containing the -636bp, -619bp, -586bp, -569bp truncations revealed that GUS
activity was located in the phloem. Expression became non-specific in transgenics containing the -547bp, -524bp, -505bp, -481 bp, and -452bp truncations, suggesting that a negative regulatory element, repressing expression in non-phloem cell types, exists between -569 and -54 7bp.
The ability of the -569 to -547bp region to bind a putative repressor protein
was investigated in electrophoretic mobility shift assays (EMS As) using specific
oligonucleotides. A MYB core sequence (CTGTTA), which is disrupted in the -
54 7bp truncation construct, was located and EMSAs revealed that an oligonucleotide containing the MYB core and 44bp of flanking sequence bound rape leaf nuclear proteins and formed two specific complexes of reduced mobility. These specific complexes were designated B 1 and B2.
Northern analysis identified developmental stages when extA was expressed;
the extA transcript is absent in mature laminae and present to high levels in mature petiole. B1 and B2 complexes were only formed with nuclear protein extracts from mature B. napus lamina and not with extracts from mature petiole, implying that the complexes consist of repressor proteins, preventing extA expression in the lamina.
Different regions of the NRR formed additional complexes with nuclear
proteins extracted from rape leaves, indicating that the NRR is functionally
composite.