Analysis of Promoter Activity for the Gene Encoding Pyruvate Orthophosphate Dikinase in Stably Transformed C4Flaveria Species1

Abstract

The C 4 enzyme pyruvate orthophosphate dikinase is encoded by a single gene, Pdk, in the C 4 plant Flaveria trinervia. This gene also encodes enzyme isoforms located in the chloroplast and in the cytosol that do not have a function in C 4 photosynthesis. Our goal is to identify cis-acting DNA sequences that regulate the expression of the gene that is active in the C 4 cycle. We fused 1.5 kb of a 5 flanking region from the Pdk gene, including the entire 5 untranslated region, to the uidA reporter gene and stably transformed the closely related C 4 species Flaveria bidentis. ␤-Glucuronidase (GUS) activity was detected at high levels in leaf mesophyll cells. GUS activity was detected at lower levels in bundle-sheath cells and stems and at very low levels in roots. This lower-level GUS expression was similar to the distribution of mRNA encoding the nonphotosynthetic form of the enzyme. We conclude that cis-acting DNA sequences controlling the expression of the C 4 form in mesophyll cells and the chloroplast form in other cells and organs are colocated within the same 5 region of the Pdk gene.

Carbonic anhydrase and the molecular evolution of C4 photosynthesis

“…It was proposed that the evolution of the C4 Flaveria chloroplastic PPDK isoform occurred through the acquisition of an alternative promoter region that directs high levels of mesophyllspecific expression of the gene (Monson 1999(Monson , 2003. Flaveria bidentis plants stably transformed with a construct encoding the 1.5 kb region flanking the 5′-end of the F. trinervia (C4) Pdk gene fused to the b-glucuronidase (GUS) reporter gene showed high levels of GUS expression in leaf mcs, lending support to the models (Rosche et al 1998).…”

The Regulation of Gene Expression Required for C4Photosynthesis

“…In stable transformants of F. bidentis, the region −1212 to +279 from the transcriptional start site fused to uidA led to high levels of GUS in the M but lower amounts in the BS, stems, and roots (105). These data indicate that the FbPPDK promoter mainly drives expression in M but can also generate expression in BS cells (105).…”

Loss of the Transit Peptide and an Increase in Gene Expression of an Ancestral Chloroplastic Carbonic Anhydrase Were Instrumental in the Evolution of the Cytosolic C4 Carbonic Anhydrase in Flaveria

“…CA1 and CA3 precursor polypeptides were imported into isolated pea chloroplasts, and concomitant with import was processing of the CA1 and CA3 precursors to lower molecular mass polypeptides, which were protected from externally added protease. Work with other C 4 photosynthetic enzymes from C 3 and C 4 Flaveria species has shown that alterations in gene expression patterns occurred during the evolution of the C 4 forms of the enzymes, which have resulted in their increased levels of expression and celland organ-specific expression patterns (Marshall et al, 1996;Drincovich et al, 1998;Rosche et al, 1998;Lai et al, 2002;Gowik et al, 2004). Similar modifications are anticipated to have occurred during the evolution of the gene encoding the C 4 form of b-CA.…”