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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 translation of rbcL
protein)
Observed as a doublet band
Occurs between -14 (relative to the initiator AUG ) and the end of the
processed rbcL 5’ UTR at –66
Only to RNAs corresponding to mature processed rbcL transcripts (5' UTR
terminating at –66); transcripts with longer or shorter 5’UTR sequences do
not to bind 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