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.

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.

Subunit III (Psa-F) of photosystem I reaction center of the C4 dicotyledon Flaveria trinervia.

An immunological survey of C3, C4 and C3-C4-intermediate Flaveria species showed that subunit III (PsaF) of the photosystem I reaction center (PSI-RC) is present in all these species. This was confirmed by the isolation of the gene encoding the PSI-RC subunit III (PsaF) from Flaveria trinervia, the first psaF gene to be isolated from a C4 plant. The deduced amino acid sequence showed a high degree of similarity to the corresponding protein of spinach which is a C3 species. A region of 17 hydrophobic amino acids in the C-terminal part of the F. trinervia protein was found to be especially conserved in all PsaF proteins studied so far (cyanobacteria and Chlamydomonas).

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.

PEG-mediated plastid transformation: a new system for transient gene expression assays in chloroplasts.

Evidence is presented for the introduction of functional copies of the GUS-reporter gene with plastid regulatory signals into chloroplasts after treatment of Nicotiana plumbaginifolia leaf protoplasts with PEG. GUS-activity is found in cells derived from protoplasts treated with PEG in the presence of plasmids harbouring the GUS-gene under the control of plastid promoter and terminator signals (plastid-specific reporter gene constructions). The activity is maintained after chloroplast isolation and incubation with the protease thermolysin under conditions sufficient to completely remove the much higher transient nuclear/cytoplasmic expression of a GUS-gene carrying the CaMV 35S-promoter. Likewise, GUS-activity derived from a plasmid coding for the nuclear/cytoplasmic expression of the reporter gene with a plastid transit presequence is also maintained after these procedures. These results indicate that PEG-treatment is a suitable protocol by which to introduce DNA into chloroplasts for the study of transient gene expression.

Differential biogenesis of photosystem-II in mesophyll and bundle-sheath cells of 'malic' enzyme NADP(+)-type C4 plants. A comparative protein and RNA analysis.

We have investigated the photosystem-II organization in differentiating-bundle-sheath cells of the three malate dehydrogenase (oxaloacetate decarboxylating) (NADP+)-type C4 species maize, Sorghum and Pennisetum. Using a set of nine different antisera raised against individual subunits of photosystem-II, we demonstrate that photosystem-II components constitute a substantial part of the thylakoid membranes of young bundle-sheath chloroplasts. The abundance of subunits of the photosystem-II core, i.e. the 47-and 43-kDa chlorophyll-a-binding proteins, polypeptides D1 and D2, cytochrome b559, and the 34-kDa polypeptide, varies with the developmental state of the plant. However, the levels of the 23-kDa, 16-kDa and 10-kDa extrinsic polypeptides of the water-oxidation complex are drastically reduced in bundle-sheath chloroplasts of all three species analyzed, regardless of their state of differentiation. The reduction in protein abundance is also reflected at the transcript level: only traces of the nuclear-encoded mRNAs are found in differentiating bundle-sheath cells of Sorghum, suggesting that the transcription of these genes has been switched off. Our data are compatible with the idea that the water-oxidation complex is a prime site for initiating or maintaining the process leading to photosystem-II depletion during differentiation of bundle-sheath cells.