Shozo Ohta

The promoter of rbcS in a C3 plant (rice) directs organ-specific, light-dependent expression in a C4 plant (maize), but does not confer bundle sheath cell-specific expression

The small subunit of ribulose-bisphosphate carboxylase (Rubisco), encoded by rbcS, is essential for photosynthesis in both C3 and C4 plants, even though the cell specificity of rbcS expression is different between C3 and C4 plants. The C3 rbcS is specifically expressed in mesophyll cells, while the C4 rbcS is expressed in bundle sheath cells, and not mesophyll cells. Two chimeric genes were constructed consisting of the structural gene encoding β-glucuronidase (GUS) controlled by the two promoters from maize (C4) and rice (C3) rbcS genes. These constructs were introduced into a C4 plant, maize. Both chimeric genes were specifically expressed in photosynthetic organs, such as leaf blade, but not in non-photosynthetic organs. The expressions of the genes were also regulated by light. However, the rice promoter drove the GUS activity mainly in mesophyll cells and relatively low in bundle sheath cells, while the maize rbcS promoter induced the activity specifically in bundle sheath cells. These results suggest that the rice promoter contains some cis-acting elements responding in an organ-pecific and light-inducible regulation manner in maize but does not contain element(s) for bundle sheath cell-specific expression, while the maize promoter does contain such element(s). Based on this result, we discuss the similarities and differences between the rice (C3) and maize (C4) rbcS promoter in terms of the evolution of the C4 photosynthetic gene.

Cold stability of pyruvate, orthophosphate dikinase of Flaveria brownii.

The nucleotide sequences of the complementary DNA of pyruvate, Pi dikinase (PPDK) from Flaveria bidentis, a C4 plant which possesses a cold-sensitive form of PPDK, and Flaveria brownii, a ‘C4-like’ plant which possesses a cold-tolerant form of PPDK, were determined. PPDK was isolated from the leaves of both Flaveria species and purified and the N-terminal amino acid sequences characterised. Together with a maize PPDK cDNA, cDNA inserts which code for the mature form of PPDK of F. bidentis and of F. brownii were expressed in bacteria and the cold sensitivity of the expressed PPDK studied. The cold sensitivity of the PPDK expressed in bacteria mimics the cold sensitivity of PPDK found in vivo in all three plant species. This study indicates that the cold sensitivity of plant PPDK is controlled by the primary structure of the enzyme.

Binding of cell type-specific nuclear proteins to the 5′-flanking region of maize C4 phosphoenolpyruvate carboxylase gene confers its differential transcription in mesophyll cells.

C4-type phosphenolpyruvate carboxylase (C4PEPC) acts as a primary carbon assimilatory enzyme in the C4 photosynthetic pathway. The maize C4PEPC gene (C4Ppc1) is specifically expressed in mesophyll cells (MC) of light-grown leaves, but the molecular mechanism responsible for its cell type-specific expression has not been characterized. In this study, we introduced a chimeric maize C4Ppc1 5′-flanking region/β-glucuronidase (GUS) gene into maize plants by Agrobacterium-mediated transformation. Activity assay and histochemical staining showed that GUS is almost exclusively localized in leaf MC of transgenic maize plants. This observation suggests that the introduced 5′ region of maize C4Ppc1 contains the necessary cis element(s) for its specific expression in MC. Next, we investigated whether the 5′ region of the maize gene interacts with nuclear proteins in a cell type-specific manner. By gel shift assays with nuclear extracts prepared from MC or bundle sheath cells (BSC), cell type-specific DNA-protein interactions were detected: nuclear factors PEPIb and PEPIc are specific to MC whereas PEPIa and PEPIIa are specific to BSC. Light alters the binding activity of these factors. These interactions were not detected in the assay with nuclear extract prepared from root, or competed out by oligonucleotides corresponding to the binding sites for the maize nuclear protein, PEP-I, which is known to bind specifically to the promoter region of C4Ppc1. The results suggest that novel cell type-specific positive and negative nuclear factors bind to the maize C4Ppc1 5′-flanking region and regulate its differential transcription in MC in a light-dependent manner.

Identification of the amino acid residues responsible for cold tolerance in Flaveria brownii pyruvate,orthophosphate dikinase

Pyruvate,orthophosphate dikinase (PPDK), an enzyme important in C4 photosynthesis, is typically a cold‐sensitive enzyme. However, a cold‐tolerant form of the enzyme has been isolated from the leaves of Flaveria brownii. Using an Escherichia coli expression system and the PPDK cDNAs from F. brownii (cold‐tolerant), F. bidentis (cold‐sensitive) and maize (intermediate cold tolerance), site‐directed mutagenesis studies indicated that as few as three amino acids residues (of 880 residues) strongly influence the cold sensitivity of Flaveria PPDK. Gel filtration analysis of the PPDK expressed in E. coli showed that subunit association and cold tolerance are closely linked.

High-level expression of cold-tolerant pyruvate, orthophosphate dikinase from a genomic clone with site- directed mutations in transgenic maize

Pyruvate, orthophosphate dikinase (PPDK) is a key enzyme in the C4 photosynthetic pathway of maize. To improve the cold tolerance of the enzyme in maize, we designed two genomic sequence-based constructs in which the carboxy-terminal region of the enzyme was modified to mimic the amino acid sequence of the cold-tolerant PPDK of Flaveria brownii (Asteraceae). A large amount of PPDK was found to have accumulated in the leaves of many of the maize plants transformed with one of these constructs – that which introduced 17 amino acid substitutions without any alteration of the exon-intron structure – although there was a wide range of variation in the amount of PPDK among the separate plants. In contrast, the production was much less in maize transformed with the second construct in which a cDNA fragment for the same carboxy-terminal region was inserted. The specific activity of PPDK in the plants transformed with the gene with the amino acid substitutions was inversely correlated with the amount of enzyme in the leaves. In addition, the activity of the cold-tolerant recombinant enzyme was judged to be regulated by the PPDK regulatory protein, similar to that of the native PPDK. The cold tolerance of PPDK in crude leaf extracts was greatly improved in plants that produced a large amount of the engineered PPDK. The photosynthetic rate at 8°C increased significantly (by 23%, p<0.05), but there was no obvious effect at higher temperatures. These results support the hypothesis that PPDK is one of the limiting factors in the C4 photosynthesis of maize under cold conditions.