Differences in Transcriptional Regulatory Mechanisms Functioning for Free Lysine Content and Seed Storage Protein Accumulation in Rice Grain

Lysine is the most defi cient 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 fi nger (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 signifi cant 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.