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 ÿ570 and a distal part from ÿ1566 to ÿ2141, which are necessary but also sufficient for high mesophyll-specific expression of the b-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.

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 C3 plants, GDC can be found in all photosynthetic cells, whereas in leaves of C3-C4 intermediate and C4 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 C4 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 C4 species Flaveria trinervia. Expression of a promoter-reporter gene fusion in transgenic plants of the transformable C4 species Flaveria bidentis (C4) 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 C4-like spatial activity also in the C3 plant Arabidopsis (Arabidopsis thaliana), indicating that a mechanism for bundle-sheath-specific expression is also present in this C3 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 C4 context.

The function of the C4 mechanism of photosynthesis depends on the strict compartmentation of the enzymes involved. Here, we investigate the regulatory mechanisms that ensure the mesophyll-specific expression of the C4 isoform of phosphoenolpyruvate carboxylase. We show that 2 kb of the 5[prime] flanking region of the Flaveria trinervia C4 PpcA1 gene is sufficient to direct mesophyll-specific expression of the [beta]-glucuronidase reporter gene in transgenic F. bidentis (C4) plants. In young leaves of seedlings, the activity of this promoter is dependent on the developmental stage of the mesophyll cells. It is induced in a basipetal fashion (leaf tip to base) during leaf development. The promoter region of the orthologous nonphotosynthetic Ppc gene of F. pringlei (C3) induces reporter gene expression mainly in the vascular tissue of leaves and stems as well as in mesophyll cells of transgenic F. bidentis plants. Our experiments demonstrate that during the evolution of the C4 Flaveria species, cis-acting elements of the C4 Ppc gene must have been altered to achieve mesophyll-specific expression.

The function of the C4 mechanism of photosynthesis depends on the strict compartmentation of the enzymes involved. Here, we investigate the regulatory mechanisms that ensure the mesophyll-specific expression of the C4 isoform of phosphoenolpyruvate carboxylase. We show that 2 kb of the 5' flanking region of the Flaveria trinervia C4 PpcA7 gene is sufficient to direct mesophyll-specific expression of the P-glucuronidase reporter gene in transgenic F. bidentis (C, ) plants. In young leaves of seedlings, the activity of this promoter is dependent on the developmental stage of the mesophyll cells. It is induced in a basipetal fashion (leaf tip to base) during leaf development. The promoter region of the orthologous nonphotosynthetic Ppc gene of F. pringlei (CJ induces reporter gene expression mainly in the vascular tissue of leaves and stems as well as in mesophyll cells of transgenic F. bidentis plants. Our experiments demonstrate that during the evolution of the C4 Flaveria species, cis-acting elements of the C4 Ppc gene must have been altered to achieve mesophyll-specific expression.

Long-term effects of herbicides on the photosynthetic membrane system were investigated. For this purpose the unicellular alga Bumilleriopsis filiformis was grown in liquid culture in the presence of sublethal concentrations of diuron, atrazine, metribuzin, and some substituted pyridazinones. This allowed reproducible growth conditions, exact dosing of herb'cides, and also the preparation of functionally active cell-free photosynthetic systems. The electron transport system of cells grown in the presence of ureas and triazine derivatives is not impaired at all, wherea scultivation with pyridazinones damages their photosynthetic redox system. In addition, a strong bleaching effect is observed, whereas the first group of herbicides enhance chlorophyll formation.Atrazin, M««riS4iEir uJWI

BackgroundThe key enzymes of photosynthetic carbon assimilation in C4 plants have evolved independently several times from C3 isoforms that were present in the C3 ancestral species. The C4 isoform of phosphoenolpyruvate carboxylase (PEPC), the primary CO2-fixing enzyme of the C4 cycle, is specifically expressed at high levels in mesophyll cells of the leaves of C4 species. We are interested in understanding the molecular changes that are responsible for the evolution of this C4-characteristic PEPC expression pattern, and we are using the genus Flaveria (Asteraceae) as a model system. It is known that cis-regulatory sequences for mesophyll-specific expression of the ppcA1 gene of F. trinervia (C4) are located within a distal promoter region (DR).ResultsIn this study we focus on the proximal region (PR) of the ppcA1 promoter of F. trinervia and present an analysis of its function in establishing a C4-specific expression pattern. We demonstrate that the PR harbours cis-regulatory determinants which account for high levels of PEPC expression in the leaf. Our results further suggest that an intron in the 5' untranslated leader region of the PR is not essential for the control of ppcA1 gene expression.ConclusionThe allocation of cis-regulatory elements for enhanced expression levels to the proximal region of the ppcA1 promoter provides further insight into the regulation of PEPC expression in C4 leaves.