During our research programs devoted to understanding the regulatory mechanisms responsible for zein gene expression in maize endosperms, we have shown that the role of transcriptional activator Opaque-2 (O2) is not only confined in controlling the expression of the 22-kDa zein gene family (Motto et al., in B.A. Larkins and I.K. Vasil eds., Cellular and Molecular Biology of Plant Seed Development, pp. 479-522, 1997). In fact, the O2 protein can transiently activate the expression of its own promoter (Lohmer et al., EMBO J. 10:617-624, 1991) and regulates directly or indirectly a number of other, non storage protein genes. These include b-32, encoding a type I ribosome-inactivating protein (Maddaloni et al., J. Genet & Breed. 45: 377-380, 1991), and b-70, encoding a heat shock protein 70 analogue, possibly acting as a chaperonin during protein body formation (Marocco et al., Plant Cell 3:507, 515, 1991). O2 also regulates the enzyme levels of LKR (lysine-ketoglutarate reductase) (Brochetto-Braga et al., Plant Physiol. 8:1139-1147, 1992), ASK1 (aspartate kinase1) (Azevedo et al., Plant Physiol. 108:103, 1995), and cyPPDK1 (cytosolic orthophosphate dikinase1) (Maddaloni et al., Mol. Gen. Genet. 250:647-654, 1996).
To assess the degrees to which changes in transketolase (TKS) protein
synthesis in maize endosperms are reflected at the mRNA level, the relative
abundance of TKS transcripts was examined by Northern blot analysis. Poly(A)+
mRNA was isolated from wild-type and o2 endosperms harvested at
15 days after pollination, size fractionated and immobilized onto membrane
filters. The filters were hybridized and washed under stringent conditions
with cDNA probes for the Tks gene from sorghum, provided by P. Westhoff
(Heinrich-Heine Universität, Dusseldof, Germany). The results clearly
demonstrated that TKS mRNA is more abundant (4-5 fold) in wild-type endosperms
than in the o2 endosperms. The O2 protein is, therefore, indeed
involved in the regulation of TKS gene expression in maize endosperm. The
same filters were checked for uniformity of loading by rehybridizing with
a specific probe for the ubiquitin gene which, in our hands, is not affected
by the o2 mutation. This observation is of particular interest due
to the fact that PPDK and TSK enzymatic activities are involved in the
synthesis of phosphoenolpyruvate and erythrose 4-phosphate, respectively;
these metabolites are the direct precursors of heptulosonate 7-phosphate,
the first compound in the shikimate pathway. This pathway is currently
receiving a great deal of interest due to its cardinal importance in the
metabolic fluxes in the plant (reviewed in Weaver and Herrmann, Trends
in Plant Science 9:346-3351, 1997). Similarly to PPDK, TSK mRNA is also
modulated by the nitrogen compounds present in the growing media.
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