The Vpp1, Esr6a, Esr6b and OCL4 promoters are active in distinct domains of maize endosperm

Abstract

Maize endosperm is both a crucial contributor to world nutrition and a model system for plant developmental biology. For both aspects promoters with expression zones restricted to particular domains of the endosperm would be a valuable tool. The β-glucuronidase ( Gus) reporter gene was fused to upstream fragments of three genes whose expression is limited to specific domains of the endosperm: Vacuolar pyrophosphatase 1 ( Vpp1) encoding an H+ translocating vacuolar pyrophosphatase and expressed in the aleurone layer, Embryo surrounding region 6 ( Esr6) encoding a defensin-like protein and expressed specifically in the embryo surrounding region (ESR) and Outer cell layer 4 ( OCL4) encoding a transcription factor of the homeo domain-leucine zipper IV (HD-ZIP IV) family and expressed in the aleurone layer. The sequence analysis of the upstream fragments revealed several putative cis elements conserved either with the rice orthologues or with other maize genes sharing the same expression domain. The phenotypic characterisation of transgenic promoter- Gus lines revealed for Vpp1 a delay in the onset of GUS activity, as compared to in situ data, and a dynamic pattern shifting from the adaxial to the abaxial side of the aleurone layer. The two closely related genes Esr6a and Esr6b discovered during the cloning process diverged strongly in their upstream sequences but showed GUS signals very similar to each other and to the in situ signal: a strong, ESR-specific GUS signal at 8 days after pollination (DAP) and 16 DAP, which disappeared at 22 DAP. While Esr6b was likely the stronger promoter, a point-like signal outside the ESR on the abgerminal side at the junction of the basal endosperm transfer layer (BETL) and the aleurone layer was only found in prEsr6a- Gus plants. The GUS signal of pr OCL4- Gus plants only partially reflected the in situ data; whereas OCL4 in situ signal was seen in the epidermis of male and female flowers, embryos and endosperm, prOCL4- GUS signal was only observed in female flowers and endosperm where it confirmed the high specificity for the outer cell layer and exhibited a dynamic pattern similar to Vpp1. While the upstream fragment used likely contained only some but not all the cis elements governing OCL4 expression, the fortuitously obtained truncated promoter may be of greater biotechnological interest than the complete promoter because of its more restricted expression zone that still includes the highly interesting aleurone layer.