Nitrogen and hormonal responsiveness of the 22 kDa alpha-zein and b-32 genes in maize endosperm is displayed in the absence of the transcriptional regulator Opaque-2.

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The maize (Zea mays L.) b-ZIP transcriptional activator Opaque-2(O2) regulates the synthesis of major endosperm proteins. In the o2 homozygote, 22 kDa zein prolamins and the b-32 ribosome-inactivating protein are greatly reduced in level. An in vitro endosperm culture system has been studied in which o2 endosperm synthesizes 22 kDa zein and b-32 in response to nitrogen supplements. An increase in 22 kDa zein mRNA concentration is also seen, implying an effect at the level of transcription or differential RNA turnover. The nitrogen-dependent induction of 22 kDa zein synthesis in cultured o2 endosperm was further investigated by analysing transient expression of reporter constructs. The highest response to nitrogen was exhibited by the intact 22 kDa zein promoter. Removal of individual O2 binding sites either reduced or increased overall promoter activity, but always decreased the nitrogen-dependent stimulation of activity. This effect was observed equally in wild-type and o2 mutant endosperm. It is concluded that a factor other then O2 is responsible for activating the 22 kDa zein promoter under high-nitrogen culture conditions. Despite its occurrence in the absence of O2 protein, the nitrogen response is mediated through binding at O2 binding sites. An induction of 22 kDa zein and b-32 synthesis in cultured o2 endosperm could also be achieved on nitrogen-free media by the addition of abscisic acid or methyl jasmonate.