Evidence is provided that plant transcription factors of the ABA response element binding protein (AREBP) class are phosphorylated by sucrose non-fermenting-1related protein kinase-1 (SnRK1) and a calcium-dependent protein kinase at highly conserved target sites. Two target sites for SnRK1 were identified in AREBPs using a specially developed motif search pipeline. Peptides containing the AREBP sites were phosphorylated by purified SnRK1 in vitro and by calcium-dependent and calcium-independent activities present in soluble protein extracted from Arabidopsis seedlings grown in liquid culture. Most (69-77%) of the calcium-independent phosphorylation of these peptides was removed by immunoprecipitation using anti-SnRK1 antisera, linking this activity unambiguously to SnRK1. It is possible that the protein kinase responsible for the calcium-dependent activity was SnRK3, a protein kinase that is related to SnRK1 and which has similar requirements for target site recognition. However, the involvement of other calcium-dependent protein kinases cannot be ruled out.

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A cDNA clone encoding the gamma-zein protein of maize was expressed in developing grain of barley using the starchy endosperm cell-specific promoter from the wheat Glu-1D-1 (HMW subunit 1Dx5) gene. Seven transgenic lines were recovered from 226 bombarded immature embryos, of which two were sterile and four tetraploid, while five were shown to express the gamma-zein protein based on western blotting. Southern blot analysis showed the presence of between about three and twelve transgene insertions. Detailed comparative studies of five null and five homozygous transformed sub-lines from transgenic line A showed that gamma-zein accounted for over 4% of the total prolamin fraction, corresponding to about 1.9% of the total grain N. Comparison of the proteins present in the gel protein fraction demonstrated that the gamma-zein was incorporated into polymers, as in maize. However, there was no effect on grain hardness measured using the Perten Single Kernel Characterisation System or on the vitreousness measured by visual inspection. This contrasts with the situation in maize where a clear association with vitreousness has been reported.