--J. Thom, J. Bailey-Serres and M. Freeling
The three cytosolic 6-PGD isozymes, PGD1 . PGD1, PGD1 . PGD2, and PGD2 . PGD2, are encoded by Pgd1 and Pgd2 (Phenotype and genetic analysis provided by Stuber and Goodman, Maydica 29: 453-471, 1984; C. Stuber per. comm.). We resolved 6-PGD dimer banding patterns in a number of tissues and organs by native polyacrylamide gel electrophoresis (Sachs and Freeling, Mol. Gen. Genet. 161:111-115, 1978), followed by PGD activity staining(0.2mg/ml NADP+, 0.2mg/ml NBT, 0.02mg/ml PMS, 1mg/ml Na3 6-phosphogluconate, 0.05 M Tris (pH 7.5), at RT for ~ 60').
Our analysis suggests that the relative steady state levels of 6-PGD isozyme dimers exhibit organ and tissue specificity. When comparing the two heterodimers, we observed the PGD2 . PGD2 to be more abundant relative to PGD1 . PGD1 in pollen, root, coleoptile, immature scutellum, and quiescent embryo axis. However, in the milky endosperm we observed the two homodimers, PGD1 . PGD1 and PGD2 . PGD2, to be present in equal amounts while the heterodimer, PGD1 . PGD2, was approximately fourfold more abundant.
In situ staining of 6-PGD activity in B73 and two Pgd2-5 null families from our Robertson's Mutator stock confirmed specific expression of Pgd2-5 in scutella. In B73, 6-PGD stained evenly throughout the scutella whereas scutella from our Pgd2-5 null homozygotes failed to stain, except for cells associated with the preprovascular bundles. This result suggests that Pgd1-3.8 expression is localized to the preprovascular bundles of the scutella. In the future, we hope to determine the precise distribution of the 6-PGD isozyme dimers within the plant tissue and organs.
At the moment, we are in the process of determining whether our two
Pdg2-5 null lines originated via Mu-induced mutations or
pre-existed in our Robertson's Mutator lines.
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