Monika Streubel

cis-Regulatory Elements for Mesophyll-Specific Gene Expression in the C4 Plant Flaveria trinervia, the Promoter of the C4 Phosphoenolpyruvate Carboxylase Gene

C4 photosynthesis depends on the strict compartmentalization of CO2 assimilatory enzymes. cis-regulatory mechanisms are described that ensure mesophyll-specific expression of the gene encoding the C4 isoform of phosphoenolpyruvate carboxylase (ppcA1) of the C4 dicot Flaveria trinervia. To elucidate and understand the anatomy of the C4 ppcA1 promoter, detailed promoter/reporter gene studies were performed in the closely related C4 species F. bidentis, revealing that the C4 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 β-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 C3 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.

Differential accumulation of plastid transcripts encoding photosystem II components in the mesophyll and bundle-sheath cells of monocotyledonous NADP-malic enzyme-type C4 plants

As a first step in understanding the differential expression of the plastid-encoded photosystem II (PSII) genes in mesophyll and bundle-sheath cells, we have used RNA blotting techniques to investigate the transcript patterns of these genes in three NADP-malic enzymetype C4 species: Zea mays L., Sorghum bicolor (L.) Moench and Pennisetum americanum (L.) Leeke. Our comparison showed that in all three species the relative levels of transcripts encoding PSII proteins were diminished in bundle-sheath cells. No major differences, either in abundance or in the processing pathways, could be detected for transcripts encoding subunits of the PSI and ATP-synthase complexes. The transcript profiles of the psbB and psbD/C transcription units were particularly striking. These operons were of heterogeneous composition, i.e. they encode PSII subunits as well as proteins or RNAs which are involved in different functional entities. The transcript patterns of the psbB and psbD/C transcription units were complex and characterized by multiple, partially overlapping RNAs. Our analysis showed that the relative levels of the oligocistronic PSII transcripts derived from these transcription units with the exception of psbH were selectively reduced in bundlesheath cells. In contrast, RNAs carrying the non-PSII components were present in similar quantities in the two cell types. The data demonstrate that segmental RNAs within one single transcription unit can accumulate to different degrees. Regulatory mechanisms which may explain this expression behaviour are discussed.

Evolution and Function of a cis-Regulatory Module for Mesophyll-Specific Gene Expression in the C4 Dicot Flaveria trinervia

C4 photosynthesis presents a sophisticated integration of two complementary cell types, mesophyll and bundle sheath cells. It relies on the differential expression of the genes encoding the component enzymes and transporters of this pathway. The entry enzyme of C4 photosynthesis, phosphoenolpyruvate carboxylase (PEPC), is found exclusively in mesophyll cells, and the expression of the corresponding gene is regulated at the transcriptional level. In the C4 dicot Flaveria trinervia, the mesophyll-specific expression of the C4 PEPC gene (ppcA) depends on a 41-bp segment in the distal promoter region referred to as MEM1 (for mesophyll expression module1). Here, we show that a MEM1 sequence found in the orthologous ppcA gene from the C3 species Flaveria pringlei is not able to direct mesophyll-specific gene expression. The two orthologous MEM1 sequences of F. pringlei and F. trinervia differ at two positions, a G-to-A exchange and the insertion of the tetranucleotide CACT. Changes at these two positions in the C3 MEM1 sequence were necessary and sufficient to create a mesophyll-specificity element during C4 evolution. The MEM1 of F. trinervia enhances mesophyll expression and concomitantly represses expression in bundle sheath cells and vascular bundles.

The molecular basis of C4 photosynthesis in sorghum: isolation, characterization and RFLP mapping of mesophyll- and bundle-sheath-specific cDNAs obtained by differential screening

C4 photosynthesis depends upon the strict compartmentalization of the CO2-assimilatory enzymes of the C4 and Calvin cycle in two different cell types, mesophyll and bundle-sheath cells. A differential accumulation is also observed for enzymes of other metabolic pathways, and mesophyll and bundle-sheath chloroplasts of NADP-malic enzyme type C4 plants differ even in their photosynthetic electron transport chains. A large number of studies indicate that this division of labour between mesophyll and bundle-sheath cells is the result of differential gene expression. To investigate the extent of this differential gene expression and thus gain insight into the genetic basis of C4 photosynthesis, genes that are differentially expressed in the mesophyll and bundle-sheath cells were catalogued in the NADP-malic enzyme type C4 grass Sorghum bicolor. A total of 58 cDNAs were isolated by differential screening. Using a tenfold difference in transcript abundance between mesophyll and bundle-sheath cells as a criterion, 25 cDNAs were confirmed to encode mesophyll-specific gene sequences and 8 were found to encode bundle-sheath-specific sequences. Eight mesophyll-specific cDNAs showed no significant similarities within GenBank and may therefore represent candidates for the elucidation of hitherto unknown functions in the differentiation of mesophyll and bundle-sheath cells. The chromosomal location of 50 isolated cDNAs was determined by RFLP mapping using an interspecific sorghum cross.

Evolution of C4 Photosynthesis in the Genus Flaveria: Establishment of a Photorespiratory CO2 Pump

The establishment of the photorespiratory CO2 pump is thought to be an early and essential step in the evolution of C4 photosynthesis. This article provides experimental evidence that the establishment of this pump in the genus Flaveria was facilitated by the differential expression of Gly decarboxylase P-protein genes. C4 photosynthesis is nature’s most efficient answer to the dual activity of ribulose-1,5-bisphosphate carboxylase/oxygenase and the resulting loss of CO2 by photorespiration. Gly decarboxylase (GDC) is the key component of photorespiratory CO2 release in plants and is active in all photosynthetic tissues of C3 plants, but only in the bundle sheath cells of C4 plants. The restriction of GDC to the bundle sheath is assumed to be an essential and early step in the evolution of C4 photosynthesis, leading to a photorespiratory CO2 concentrating mechanism. In this study, we analyzed how the P-protein of GDC (GLDP) became restricted to the bundle sheath during the transition from C3 to C4 photosynthesis in the genus Flaveria. We found that C3 Flaveria species already contain a bundle sheath–expressed GLDP gene in addition to a ubiquitously expressed second gene, which became a pseudogene in C4 Flaveria species. Analyses of C3-C4 intermediate Flaveria species revealed that the photorespiratory CO2 pump was not established in one single step, but gradually. The knowledge gained by this study sheds light on the early steps in C4 evolution.

Primary structure of the photosynthetic pyruvate orthophosphate dikinase of the C3 plant Flaveria pringlei and expression analysis of pyruvate orthophosphate dikinase sequences in C3, C3-C4 and C4 Flaveria species.

We have isolated full-size cDNA sequences encoding the photosynthetic isoform of pyruvate orthophosphate dikinase (PPDK) of the C3 plant Flaveria pringlei. The encoded protein shares 96% identical amino acid residues with the C4 isoform of PPDK in the C4 species F. trinervia. The differing amino acid residues are evenly distributed along the polypeptide chain. Genomic Southern analysis of photosynthetic PPDK sequences in F. pringlei (C3), F. chloraefolia (C3–C4), F. linearis (C3–C4), F. floridana (C3–C4), F. brownii (C4-like) and F. trinervia (C4) reveals a simple hybridization pattern which is suggestive of a single gene. Northern hybridization experiments show that the abundance of PPDK transcripts in leaves correlates with the degree of C4 characteristics expressed in the various photosynthetic types analysed. This finding demonstrates that the increase in expression levels must have played a crucial role in evolving the C4-PPDK gene in the genus Flaveria.

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 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.

Regulation of the Photorespiratory GLDPA Gene in C4 Flaveria: An Intricate Interplay of Transcriptional and Posttranscriptional Processes

This work finds that two promoters in tandem cause strong expression of the Gly decarboxylase P-subunit gene of the C4 dicot Flaveria trinervia, GLDPA, in the bundle sheath and additionally ensure weak expression in the mesophyll. This allows for large amounts of GLDPA in the bundle sheath for photorespiration and low quantities in all biosynthetic cells for C1 metabolism. The mitochondrial Gly decarboxylase complex (GDC) is a key component of the photorespiratory pathway that occurs in all photosynthetically active tissues of C3 plants but is restricted to bundle sheath cells in C4 species. GDC is also required for general cellular C1 metabolism. In the Asteracean C4 species Flaveria trinervia, a single functional GLDP gene, GLDPA, encodes the P-subunit of GDC, a decarboxylating Gly dehydrogenase. GLDPA promoter reporter gene fusion studies revealed that this promoter is active in bundle sheath cells and the vasculature of transgenic Flaveria bidentis (C4) and the Brassicacean C3 species Arabidopsis thaliana, suggesting the existence of an evolutionarily conserved gene regulatory system in the bundle sheath. Here, we demonstrate that GLDPA gene regulation is achieved by an intricate interplay of transcriptional and posttranscriptional mechanisms. The GLDPA promoter is composed of two tandem promoters, PR2 and PR7, that together ensure a strong bundle sheath expression. While the proximal promoter (PR7) is active in the bundle sheath and vasculature, the distal promoter (PR2) drives uniform expression in all leaf chlorenchyma cells and the vasculature. An intron in the 5′ untranslated leader of PR2-derived transcripts is inefficiently spliced and apparently suppresses the output of PR2 by eliciting RNA decay.

Most photorespiratory genes are preferentially expressed in the bundle sheath cells of the C4 grass Sorghum bicolor

Highlight Photorespiratory genes are expressed tissue-specific in the leaves of the C4 grass Sorghum bicolor. Most but not all of them are confined to the bundle sheath cells.

Biogenesis of Photosystem II in C3 and C4 Plants — A Model System to Study Developmentally Regulated and Cell-Specific Expression of Plastid Genes

Ordered plant development requires the coordinated, yet differential expression of functionally linked genes both in time and space. Differentiation thus is an inherent feature of all developmental processes. It is ultimately determined by the plant’s endogenous genetic programme, however, it may be initiated or modulated by external factors, with light being the most important one (1).