Evolution of C4 photosynthesis in the genus flaveria: establishment of a photorespiratory CO2 pump.

C4 photosynthesis is nature's most efficient answer to the dual activity of ribulose-1,5-bisphosphate carboxylase/oxygenase and the resulting loss of CO(2) by photorespiration. Gly decarboxylase (GDC) is the key component of photorespiratory CO(2) release in plants and is active in all photosynthetic tissues of C(3) plants, but only in the bundle sheath cells of C(4) plants. The restriction of GDC to the bundle sheath is assumed to be an essential and early step in the evolution of C(4) photosynthesis, leading to a photorespiratory CO(2) concentrating mechanism. In this study, we analyzed how the P-protein of GDC (GLDP) became restricted to the bundle sheath during the transition from C(3) to C(4) photosynthesis in the genus Flaveria. We found that C(3) Flaveria species already contain a bundle sheath-expressed GLDP gene in addition to a ubiquitously expressed second gene, which became a pseudogene in C(4) Flaveria species. Analyses of C(3)-C(4) intermediate Flaveria species revealed that the photorespiratory CO(2) pump was not established in one single step, but gradually. The knowledge gained by this study sheds light on the early steps in C(4) evolution.

The gene for the P-subunit of glycine decarboxylase from the C4 species Flaveria trinervia: analysis of transcriptional control in transgenic Flaveria bidentis (C4) and Arabidopsis (C3).

Glycine decarboxylase (GDC) plays an important role in the photorespiratory metabolism of plants. GDC is composed of four subunits (P, H, L, and T) with the P-subunit (GLDP) serving as the actual decarboxylating unit. In C(3) plants, GDC can be found in all photosynthetic cells, whereas in leaves of C(3)-C(4) intermediate and C(4) species its occurrence is restricted to bundle-sheath cells. The specific expression of GLDP in bundle-sheath cells might have constituted a biochemical starting point for the evolution of C(4) photosynthesis. To understand the molecular mechanisms responsible for restricting GLDP expression to bundle-sheath cells, we performed a functional analysis of the GLDPA promoter from the C(4) species Flaveria trinervia. Expression of a promoter-reporter gene fusion in transgenic plants of the transformable C(4) species Flaveria bidentis (C(4)) showed that 1,571 bp of the GLDPA 5' flanking region contain all the necessary information for the specific expression in bundle-sheath cells and vascular bundles. Interestingly, we found that the GLDPA promoter of F. trinervia exhibits a C(4)-like spatial activity also in the C(3) plant Arabidopsis (Arabidopsis thaliana), indicating that a mechanism for bundle-sheath-specific expression is also present in this C(3) species. Using transgenic Arabidopsis, promoter deletion studies identified two regions in the GLDPA promoter, one conferring repression of gene expression in mesophyll cells and one functioning as a general transcriptional enhancer. Subsequent analyses in transgenic F. bidentis confirmed that these two segments fulfill the same function also in the C(4) context.

cis-Regulatory elements for mesophyll-specific gene expression in the C4 plant Flaveria trinervia, the promoter of the C4 phosphoenolpyruvate carboxylase gene.

C(4) photosynthesis depends on the strict compartmentalization of CO(2) assimilatory enzymes. cis-regulatory mechanisms are described that ensure mesophyll-specific expression of the gene encoding the C(4) isoform of phosphoenolpyruvate carboxylase (ppcA1) of the C(4) dicot Flaveria trinervia. To elucidate and understand the anatomy of the C(4) ppcA1 promoter, detailed promoter/reporter gene studies were performed in the closely related C(4) species F. bidentis, revealing that the C(4) promoter contains two regions, a proximal segment up to -570 and a distal part from -1566 to -2141, which are necessary but also sufficient for high mesophyll-specific expression of the beta-glucuronidase reporter gene. The distal region behaves as an enhancer-like expression module that can direct mesophyll-specific expression when inserted into the ppcA1 promoter of the C(3) plant F. pringlei. Mesophyll expression determinants were restricted to a 41-bp segment, referred to as mesophyll expression module 1 (Mem1). Evolutionary and functional studies identified the tetranucleotide sequence CACT as a key component of Mem1.