Two Genes Encode Highly Similar Chloroplastic NADP-Malic Enzymes in Flaveria (Implications for the Evolution of C4 Photosynthesis)

Abstract

To gain an understanding of the molecular events underlying the evolution of C, photosynthesis, we have undertaken a detailed study of the NADP-malic enzyme gene family in C, and C, species of Haveria. Three genomic clones from the C, species Haveria bidentis were characterized and found to encode two highly similar chloroplastic forms of NADP-malic enzyme, termed ME1 and ME2. Genomic Southern blotting with gene-specific probes showed that both Me7 and Me2 are found in Haveria trinervia (C,) and fiaveria pringlei (C,) as well as in F. bidentis. Northern blots demonstrated that M e l expression in leaves parallels the degree of C, photosynthesis in seven Haveria species. Furthermore, whereas M e 2 was expressed at a low level in both roots and leaves of f . bidentis, M e l expression was seen only in leaves and was light-regulated. We discuss these results in the context of the evolution of C, photosynthesis in Flaveria.The C, photosynthetic pathway is a recently evolved modification of the C, pathway in which plants spatially separate the initial fixation and subsequent conversion of CO, to 3-phosphoglycerate and other carbohydrates. Through the use of this system, C, plants effectively concentrate CO, in the vicinity of Rubisco (located in bundle-sheath cells) and, thus, avoid the wasteful process of photorespiration. The efficient operation of C, photosynthesis requires the strict compartmentation of a suite of enzymes in either mesophyll or bundle-sheath cells. This intercellular compartmentation is a distinguishing feature of C, plants: a11 of the key enzymes utilized in C, photosynthesis are present in C, plants; however, their expression patterns are distinct from those seen in C, plants. It is generally accepted that C, plants evolved from C, ancestors and that the specific enzymes utilized in C, photosynthesis derived from corresponding enzymes present in the C, ancestors (Cockburn, 1983). In the evolution of C, photosynthesis, the expression programs of these ancestral proteins were Present address:

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.…”

“…Genomic DNA was isolated from leaf tissue according to the method of Marshall et al (1996), and 10-mg aliquots were digested to completion in separate reactions with EcoRI and XbaI. Genomic DNA fragments were separated by electrophoresis on a 0.8% (w/v) agarose gel and transferred to Hybond N + membranes (GE Healthcare Life Sciences) using alkaline capillary blotting (Sambrook et al, 1989).…”

“…The enzymes catalyzing the steps in the C 4 pathway are also present in C 3 plants, and several, including PEPC (Hermans and Westhoff, 1990;Kawamura et al, 1992;Gowik et al, 2006), NADP-malic enzyme (Rajeevan et al, 1991;Marshall et al, 1996;Drincovich et al, 1998;Lai et al, 2002;Tausta et al, 2002), and pyruvate orthophosphate dikinase (Rosche et al, 1994;Imaizumi et al, 1997), have been examined in the context of C 4 evolution. Many of these studies (Hermans and Westhoff, 1990;Rajeevan et al, 1991;Rosche et al, 1994;Marshall et al, 1996;Drincovich et al, 1998;Lai et al, 2002) used members of the genus Flaveria as model organisms for understanding the steps involved in the evolution of C 4 photosynthesis, because this group of closely related plants contains individual species demonstrating C 3 , C 4 , or intermediate C 3 -C 4 photosynthesis, indicating that the evolution toward C 4 photosynthesis is ongoing in this genus (McKown et al, 2005).…”

“…Many of these studies (Hermans and Westhoff, 1990;Rajeevan et al, 1991;Rosche et al, 1994;Marshall et al, 1996;Drincovich et al, 1998;Lai et al, 2002) used members of the genus Flaveria as model organisms for understanding the steps involved in the evolution of C 4 photosynthesis, because this group of closely related plants contains individual species demonstrating C 3 , C 4 , or intermediate C 3 -C 4 photosynthesis, indicating that the evolution toward C 4 photosynthesis is ongoing in this genus (McKown et al, 2005). The results from several of these studies demonstrated that the genes encoding the enzymes important in C 4 photosynthesis evolved through the duplication of ancestral C 3 genes followed by the neofunctionalization of one copy, which involved increased gene expression in specific organs and cell types (for review, see Monson, 2003).…”

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

“…A similar analysis identified two genes encoding chloroplast-localized ME isoforms in Flaveria (Marshall et al, 1996). One gene, Me1, encodes the C 4 form in the C 4 species.…”