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

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.

[1]  Taiji Kawakatsu,et al.  Cereal seed storage protein synthesis: fundamental processes for recombinant protein production in cereal grains. , 2010, Plant biotechnology journal.

[2]  H. Yasuda,et al.  Reducing Rice Seed Storage Protein Accumulation Leads to Changes in Nutrient Quality and Storage Organelle Formation1[W][OA] , 2010, Plant Physiology.

[3]  Hiroshi Yasuda,et al.  Compensation and interaction between RISBZ1 and RPBF during grain filling in rice. , 2009, The Plant journal : for cell and molecular biology.

[4]  M. Motto,et al.  Chromatin and DNA Modifications in the Opaque2-Mediated Regulation of Gene Transcription during Maize Endosperm Development[W] , 2009, The Plant Cell Online.

[5]  M. Yano,et al.  Characterization of a new rice glutelin gene GluD-1 expressed in the starchy endosperm , 2008, Journal of experimental botany.

[6]  G. Galili,et al.  Improving the Content of Essential Amino Acids in Crop Plants: Goals and Opportunities1 , 2008, Plant Physiology.

[7]  J. Vicente-Carbajosa,et al.  The maize Dof protein PBF activates transcription of γ-zein during maize seed development , 2008, Plant Molecular Biology.

[8]  Christopher P. Bonin,et al.  High-lysine corn generated by endosperm-specific suppression of lysine catabolism using RNAi. , 2007, Plant biotechnology journal.

[9]  Z. Ni,et al.  Wheat Dof transcription factor WPBF interacts with TaQM and activates transcription of an alpha-gliadin gene during wheat seed development , 2006, Plant Molecular Biology.

[10]  H. Yasuda,et al.  High accumulation of bioactive peptide in transgenic rice seeds by expression of introduced multiple genes. , 2006, Plant biotechnology journal.

[11]  Y. Onodera,et al.  Synergism between RPBF Dof and RISBZ1 bZIP Activators in the Regulation of Rice Seed Expression Genes1[W] , 2006, Plant Physiology.

[12]  Richard D. Thompson,et al.  Interaction of maize Opaque-2 and the transcriptional co-activators GCN5 and ADA2, in the modulation of transcriptional activity , 2004, Plant Molecular Biology.

[13]  F. Takaiwa,et al.  Evaluation of tissue specificity and expression strength of rice seed component gene promoters in transgenic rice. , 2004, Plant biotechnology journal.

[14]  Y. Onodera,et al.  A Rice Functional Transcriptional Activator, RISBZ1, Responsible for Endosperm-specific Expression of Storage Protein Genes through GCN4 Motif* , 2001, The Journal of Biological Chemistry.

[15]  B. Larkins,et al.  Genetic analysis of amino acid accumulation in opaque-2 maize endosperm. , 2001, Plant physiology.

[16]  P. Arruda,et al.  Regulation of lysine catabolism in higher plants. , 2000, Trends in plant science.

[17]  K. Harada,et al.  Quantitative nature of the Prolamin-box, ACGT and AACA motifs in a rice glutelin gene promoter: minimal cis-element requirements for endosperm-specific gene expression. , 2000, The Plant journal : for cell and molecular biology.

[18]  P. Arruda,et al.  The Role of Opaque2 in the Control of Lysine-Degrading Activities in Developing Maize Endosperm , 1999, Plant Cell.

[19]  I. Díaz,et al.  Barley BLZ2, a Seed-specific bZIP Protein That Interacts with BLZ1 in Vivo and Activates Transcription from the GCN4-like motif of B-hordein Promoters in Barley Endosperm* , 1999, The Journal of Biological Chemistry.

[20]  S. Toki,et al.  Iron fortification of rice seed by the soybean ferritin gene , 1999, Nature Biotechnology.

[21]  M. Mena,et al.  An endosperm-specific DOF protein from barley, highly conserved in wheat, binds to and activates transcription from the prolamin-box of a native B-hordein promoter in barley endosperm. , 1998, The Plant journal : for cell and molecular biology.

[22]  A. Suzuki,et al.  The GCN4 motif in a rice glutelin gene is essential for endosperm-specific gene expression and is activated by Opaque-2 in transgenic rice plants. , 1998, The Plant journal : for cell and molecular biology.

[23]  J. Zhu-Shimoni,et al.  Regulation of lysine catabolism through lysine-ketoglutarate reductase and saccharopine dehydrogenase in Arabidopsis. , 1997, The Plant cell.

[24]  S. Moose,et al.  A maize zinc-finger protein binds the prolamin box in zein gene promoters and interacts with the basic leucine zipper transcriptional activator Opaque2. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[25]  R. Azevedo,et al.  The enzymology of lysine catabolism in rice seeds--isolation, characterization, and regulatory properties of a lysine 2-oxoglutarate reductase/saccharopine dehydrogenase bifunctional polypeptide. , 1997, European journal of biochemistry.

[26]  M. Holdsworth,et al.  The wheat transcriptional activator SPA: a seed-specific bZIP protein that recognizes the GCN4-like motif in the bifactorial endosperm box of prolamin genes. , 1997, The Plant cell.

[27]  Richard D. Thompson,et al.  The transcriptional activatorOpaque-2 controls the expression of a cytosolic form of pyruvate orthophosphate dikinase-1 in maize endosperms , 1996, Molecular and General Genetics MGG.

[28]  P. Arruda,et al.  Purification and Characterization of the Bifunctional Enzyme Lysine-Ketoglutarate Reductase-Saccharopine Dehydrogenase from Maize , 1996, Plant physiology.

[29]  P. Arruda,et al.  The involvement of Opaque 2 on β-prolamin gene regulation in maize and Coix suggests a more general role for this transcriptional activator , 1995, Plant Molecular Biology.

[30]  R. Schmidt,et al.  Opaque-2 is a transcriptional activator that recognizes a specific target site in 22-kD zein genes. , 1992, The Plant cell.

[31]  F. Salamini,et al.  The maize regulatory locus Opaque‐2 encodes a DNA‐binding protein which activates the transcription of the b‐32 gene. , 1991, The EMBO journal.

[32]  Richard D. Thompson,et al.  The b-32 protein from maize endosperm: characterization of genomic sequences encoding two alternative central domains , 1990, Plant Molecular Biology.

[33]  F. Salamini,et al.  The O2 gene which regulates zein deposition in maize endosperm encodes a protein with structural homologies to transcriptional activators. , 1989, The EMBO journal.

[34]  E. Mertz,et al.  Mutant Gene That Changes Protein Composition and Increases Lysine Content of Maize Endosperm , 1964, Science.

[35]  Richard D. Thompson,et al.  The transcriptional activator , 1996 .

[36]  B. Burr,et al.  Maize regulatory gene opaque-2 encodes a protein with a "leucine-zipper" motif that binds to zein DNA. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[37]  H. Hayashi,et al.  Chemical composition of phloem sap from the uppermost internode of the rice plant , 1990 .