The maize O2 and PBF proteins act additively to promote transcription from storage protein gene promoters in rice endosperm cells.

A transient expression assay system was employed to investigate the possible use of the maize Opaque 2 (O2) and prolamin box binding factor (PBF) proteins as transcriptional activators of rice and wheat storage protein gene promoters. When assayed in developing rice endosperm cells, either O2 or PBF alone could increase transcription from the promoter of the rice glutelin gene, Gt1. However, mutant forms of O2 and PBF that are defective in DNA binding could not. Co-transfection with both transcriptional activators resulted in an additive increase in transactivation of the Gt1 promoter. Co-bombardment of a Gt1::GUS construct with plasmids expressing the DNA binding domains of O2 and PBF in antisense orientation resulted in a decrease of GUS expression below background levels. Similar stimulatory and additive effects of O2 and PBF could be observed on the promoters from other storage protein genes including rice globulin (Glb), prolamins (RP6 and PG5a) and a wheat glutenin (Bx7). However, responsiveness of the promoters from non-storage protein genes like rice actin and CaMV 35S to O2 and PBF was insignificant. Our results indicate that the maize O2 and PBF proteins can act singly or additively as effective stimulators of heterologous storage protein promoters in developing rice endosperm cells. These data support the use of well-characterized transcription factors from maize as an effective means of increasing the expression level of recombinant proteins in developing rice seeds.

[1]  K. Singh,et al.  Transcriptional regulation in plants: the importance of combinatorial control. , 1998, Plant physiology.

[2]  J. A. Yunes,et al.  Cooperative DNA Binding and Sequence Discrimination by the Opaque2 bZIP Factor , 1998, Plant Cell.

[3]  N. Huang,et al.  Rice expressing lactoferrin and lysozyme has antibiotic-like properties when fed to chicks. , 2002, The Journal of nutrition.

[4]  S. Nandi,et al.  Expression of human lactoferrin in transgenic rice grains for the application in infant formula , 2002 .

[5]  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.

[6]  M. Müller,et al.  The nitrogen response of a barley C-hordein promoter is controlled by positive and negative regulation of the GCN4 and endosperm box. , 1993, The Plant journal : for cell and molecular biology.

[7]  P. Arruda,et al.  Identification of a DNA-binding factor that recognizes an α-coixin promoter and interacts with a Coix Opaque-2 like protein , 2004, Plant Molecular Biology.

[8]  R. S. Conlan,et al.  Transcription activation mediated by the bZIP factor SPA on the endosperm box is modulated by ESBF-1 in vitro. , 1999, The Plant journal : for cell and molecular biology.

[9]  M. Holdsworth,et al.  In vivo footprinting of a low molecular weight glutenin gene (LMWG‐1D1) in wheat endosperm. , 1993, The EMBO journal.

[10]  O. Schabenberger,et al.  Arabidopsis CBF1 overexpression induces COR genes and enhances freezing tolerance. , 1998, Science.

[11]  S. Yanagisawa,et al.  Diversity and similarity among recognition sequences of Dof transcription factors. , 1999, The Plant journal : for cell and molecular biology.

[12]  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.

[13]  P. Busk,et al.  The bifactorial endosperm box of gamma-zein gene: characterisation and function of the Pb3 and GZM cis-acting elements. , 1998, The Plant journal : for cell and molecular biology.

[14]  B. Bowen,et al.  Dominant Negative Mutants of Opaque2 Suppress Transactivation of a 22-kD Zein Promoter by Opaque2 in Maize Endosperm Cells. , 1993, The Plant cell.

[15]  N. Huang,et al.  Expression of the REB transcriptional activator in rice grains improves the yield of recombinant proteins whose genes are controlled by a Reb-responsive promoter , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[16]  S. Yanagisawa.  A novel DNA-binding domain that may form a single zinc finger motif. , 1995, Nucleic acids research.

[17]  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.

[18]  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.

[19]  L. Kricka,et al.  Chemiluminescent reporter gene assays: sensitive detection of the GUS and SEAP gene products. , 1994, BioTechniques.

[20]  P. Quail,et al.  Ubiquitin promoter-based vectors for high-level expression of selectable and/or screenable marker genes in monocotyledonous plants , 1996, Transgenic Research.

[21]  E. Grotewold,et al.  Engineering Secondary Metabolism in Maize Cells by Ectopic Expression of Transcription Factors , 1998, Plant Cell.

[22]  Richard D. Thompson,et al.  Structural and functional analysis of an Opaque-2-related gene from sorghum , 1994, Plant Molecular Biology.

[23]  P. Arruda,et al.  The molecular and functional characterization of an Opaque2 homologue gene from Coix and a new classification of plant bZIP proteins , 2004, Plant Molecular Biology.

[24]  E. Cocking,et al.  Protoplast culture of rice (Oryza sativa L.) using media solidified with agarose , 1986 .

[25]  M. Kreis,et al.  Nucleotide sequence of a B1 hordein gene and the identification of possible upstream regulatory elements in endosperm storage protein genes from barley, wheat and maize. , 1985, Nucleic acids research.

[26]  M. Holdsworth,et al.  The maize transcription factor Opaque-2 activates a wheat glutenin promoter in plant and yeast cells , 1995, Plant Molecular Biology.

[27]  C. Schwechheimer,et al.  The regulation of transcription factor activity in plants , 1998 .

[28]  F. Katagiri,et al.  Plant transcription factors: present knowledge and future challenges. , 1992, Trends in genetics : TIG.

[29]  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.

[30]  Jesús Vicente-Carbajosa,et al.  Genome-wide comparative phylogenetic analysis of the rice and Arabidopsis Dof gene families , 2003, BMC Evolutionary Biology.

[31]  I. Díaz,et al.  Synergistic Activation of Seed Storage Protein Gene Expression in Arabidopsis by ABI3 and Two bZIPs Related to OPAQUE2* , 2003, Journal of Biological Chemistry.

[32]  J. Messing,et al.  Mutations of the 22- and 27-kD zein promoters affect transactivation by the Opaque-2 protein. , 1992, The Plant cell.

[33]  S. Goff,et al.  Transactivation of anthocyanin biosynthetic genes following transfer of B regulatory genes into maize tissues. , 1990, The EMBO journal.

[34]  E. Stockinger,et al.  Function and regulation of Arabidopsis thaliana COR (cold-regulated) genes , 1997, Acta Physiologiae Plantarum.

[35]  T. MacRae,et al.  Transcription factors and their genes in higher plants functional domains, evolution and regulation. , 1999, European journal of biochemistry.

[36]  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.

[37]  B. Burr,et al.  An arginine to lysine substitution in the bZIP domain of an opaque-2 mutant in maize abolishes specific DNA binding. , 1991, Genes & development.

[38]  R. Schmidt,et al.  bZIP Proteins Regulating Gene Expression in Maize Endosperm , 1994 .

[39]  K. Struhl,et al.  Current Protocols in Molecular Biology (New York: Greene Publishing Associates and Wiley-Interscience). Host-Range Shuttle System for Gene Insertion into the Chromosomes of Gram-negative Bacteria. , 1988 .

[40]  Kazuo Shinozaki,et al.  Improving plant drought, salt, and freezing tolerance by gene transfer of a single stress-inducible transcription factor , 1999, Nature Biotechnology.

[41]  R. Schmidt,et al.  Regulation of α-Zein Gene Expression During Maize Endosperm Development , 1994 .

[42]  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.

[43]  S. Yanagisawa.  The Dof family of plant transcription factors. , 2002, Trends in plant science.

[44]  K. Wood,et al.  Firefly luciferase gene: structure and expression in mammalian cells , 1987, Molecular and cellular biology.

[45]  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.

[46]  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.

[47]  C. Schwechheimer,et al.  PLANT TRANSCRIPTION FACTOR STUDIES. , 1998, Annual review of plant physiology and plant molecular biology.

[48]  D. Bastola,et al.  Alfin1, a novel zinc-finger protein in alfalfa roots that binds to promoter elements in the salt-inducible MsPRP2 gene , 1998, Plant Molecular Biology.

[49]  N. Huang,et al.  Evaluation of expression cassettes in developing rice endosperm using a transient expression assay , 2001 .

[50]  I. Díaz,et al.  Barley BLZ1: a bZIP transcriptional activator that interacts with endosperm-specific gene promoters. , 1998, The Plant journal : for cell and molecular biology.

[51]  Simon C Watkins,et al.  Expression and localization of human lysozyme in the endosperm of transgenic rice , 2003, Planta.

[52]  L. Nover.  Plant Promoters and Transcription Factors , 1994, Results and Problems in Cell Differentiation.

[53]  R. Rodriguez,et al.  Expression of natural antimicrobial human lysozyme in rice grains , 2002, Molecular Breeding.

[54]  R. Schmidt,et al.  OHP1: a maize basic domain/leucine zipper protein that interacts with opaque2. , 1993, The Plant cell.

[55]  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.