Differences in transcriptional regulatory mechanisms functioning for free lysine content and seed storage protein...

Lysine is the most deficient essential amino acid in cereal grains. A bifunctional lysine-degrading enzyme, lysine ketoglutarate reductase/saccharopine dehydrogenase (LKR/SDH), is one of the key regulators determining free lysine content in plants. In rice (Oryza sativa. L), a bifunctional OsLKR/SDH is predominantly present in seeds. Here, we show that OsLKR/SDH is directly regulated by major transcriptional regulators of seed storage protein (SSP) genes: the basic leucine zipper (bZIP) transcription factor (TF), RISBZ1, and the DNA-binding with one finger (DOF) transcription factor, RPBF. OsLKR/SDH was highly expressed in the aleurone and subaleurone layers of the endosperm. Mutation analyses in planta, trans-activation reporter assays in vivo and electrophorestic mobility shift assays in vitro showed that the RPBF-recognizing prolamin box (AAAG) and the RISBZ1-recognizing GCN4 motif (TGAG/CTCA) act as important cis-elements for proper expression of OsLKR/SDH like SSP genes. However, mutation of the GCN4 motif within ProOsLKR/SDH did not alter the spatial expression pattern, whereas mutation of the GCN4 motif within ProGluB-1 did alter spatial expression. Reducing either RISBZ1 or RPBF decreased OsLKR/SDH levels, resulting in an increase in free lysine content in rice grain. This result was in contrast to the fact that a significant reduction of SSP was observed only when these transcription factors were simultaneously reduced, suggesting that RISBZ1 and RPBF regulate SSP genes and OsLKR/SDH with high and limited redundancy, respectively. The same combinations of TF and cis-elements are involved in the regulation of OsLKR/SDH and SSP genes, but there is a distinct difference in their regulation mechanisms.