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C4 dicots Amaranth Flaveria Ribulose 1,5-Bisphosphate Carboxylase (Rubisco) Reactions: CO2 + H2O RuBP (5C) 2 X 3-P-Glycerate (3C) + 2 H+ Rubisco O2 + H2O RuBP (5C) Rubisco 1 X 3-P-Glycerate (3C) + 2H+ + 1 X 2-P-Glycolate (2C) Located in chloroplasts: 6 Large Subunits (LSU), 55 kDa. rbcL gene Encoded on chloroplast genome Contains substrate (CO2 and O2) binding site. 6 Small Subunits (SSU), 12-18 kDa. RbcS gene Encoded in nuclear genome as gene family Synthesized as precursor, 20 Kda, with plastid transit sequence Transported to chloroplasts Possibly for regulation and assembly How are rbcL and RbcS genes encoding Rubisco regulated in C4 plants? Regulated by leaf development? Regulated by light? Transcriptional? Post-transcriptional? A B C A B In vivo protein synthesis and mRNA levels Polysome analysis: RbcS and rbcL mRNAs are associated with polysomes in L and in D+5hr L plants RbcS and rbcL mRNAs are not associated with polysomes in D plants Regulation in response to light occurs at the level of translation initiation Rubisco rbcL and RbcS genes are regulated post-transcriptionally in C4 plants Rubisco gene expression in C4 leaves is influenced by: Light Leaf development Photosynthesis Genes encoding other C4 enzymes show independent patterns of regulation What proteins regulate translation or stability of the Rubisco mRNAs, and what cis-acting sequences are recognized by these proteins? Two approaches: Biochemical analysis (search for mRNA binding proteins) Transgenic C4 plants (Search for cis-acting regulatory sequences) p47 binding to rbcL 5'RNA: Is highly specific to rbcL 5’ RNA Only in L extracts, not in E extracts (correlates with translatio n of rbcL protein) Observed as a double t band Occurs between -14 (relative to the init iator AUG) and the end of the processed rbcL 5’ UTR at –66 Only to RNAs corresponding to mature processed rbcL transcripts (5' UTR termin ating at –66); transcrip ts with longer or shorter 5’UTR sequences do not to bin d p47 Suggests a relationship between p47 binding, transcript processing, and rbcL translation in L-plants McCormac et al., JBC 276, 2001 pSK+ to generate transcripts for in vitro translation (T3) pBI221 for Biolistic (gene gun) transient expression (CaMV) pGA482 for expression in stably transformed C4 plants (CaMV) Graduate Students: Jingliang Wang Ping Xu Amy Swec Joe Boinski John Long Amy Corey Jianxin Wang Hanz Litz Vince Ramsperger Sabrina Picinic Francois Faure Postdocs: Minesh Patel Robert Givens Mei-Hui Lin Dennis McCormac Technicians: Sejal Patel Xiaowen Jang Collaborators: John Carr (Cambridge) Bill Taylor (Canberra) Li Ping Lin (Beijing) V. James Hernandez (UB) Derek Taylor (UB) Jeremy Bruenn (UB) Mary Bisson (UB) Funding: NSF Biochemistry of gene expression International programs USDA Photosynthesis Plant genetic mechanisms Undergraduates: Jackson Buss Elizabeth Herric Theresa Flood Francois Faure Kwan Lau Eric Rydzik Paul Nordone Marina Boruk Jeff Burkwitz Janet Lam Jay Friedman Melinda Bauman Renata Mazzei Diane Watkins Matt Bernhard Peter Depowski Curtis Erickson