Oxygen Requirement and Inhibition of C4 Photosynthesis . An Analysis of C4 Plants Deficient in the C3 and C4 Cycles

The basis for O2 sensitivity of C4 photosynthesis was evaluated using a C4-cycle-limited mutant of Amaranthus edulis (a phosphoenolpyruvate carboxylase-deficient mutant), and a C3-cycle-limited transformant of Flaveria bidentis (an antisense ribulose-1,5-bisphosphate carboxylase/oxygenase [Rubisco] small subunit transformant). Data obtained with the C4-cycle-limited mutant showed that atmospheric levels of O2 (20 kPa) caused increased inhibition of photosynthesis as a result of higher levels of photorespiration. The optimal O2 partial pressure for photosynthesis was reduced from approximately 5 kPa O2 to 1 to 2 kPa O2, becoming similar to that of C3 plants. Therefore, the higher O2 requirement for optimal C4 photosynthesis is specifically associated with the C4 function. With the Rubisco-limited F. bidentis, there was less inhibition of photosynthesis by supraoptimal levels of O2 than in the wild type. When CO2 fixation by Rubisco is limited, an increase in the CO2 concentration in bundle-sheath cells via the C4 cycle may further reduce the oxygenase activity of Rubisco and decrease the inhibition of photosynthesis by high partial pressures of O2 while increasing CO2 leakage and overcycling of the C4 pathway. These results indicate that in C4 plants the investment in the C3 and C4 cycles must be balanced for maximum efficiency

Binding of cell type-specific nuclear proteins to the 5 '-flanking region of maize C-4 phosphoenolpyruvate carboxylase gene confers its differential transcription in mesophyll cells

C-4-type phosphenolpyruvate carboxylase (C4PEPC) acts as a primary carbon assimilatory enzyme in the C-4 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/beta -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

Photosynthetic Mechanisms of Weeds in Taiwan

One hundred and one species (in 36 families) of weeds on cultivated land in Taiwan were investigated for the occurrence of Kranz leaf anatomy and activities of key enzymes of C-4 photosynthesis to determine their photosynthetic mechanisms. Based on the anatomical and biochemical analyses, 75 species were found to possess the C-3 and 26 species the C-4 pathway of photosynthetic CO2 fixation. Among the 26 C-4 species, 15 species are in Gramineae, 6 in Cyperaceae, 2 each in Euphorbiaceae and Amaranthaceae, and 1 in Portulacaceae. Two C-4 species in the Gramineae, namely Digitaria radicosa (Presl) Miq. and Sporobolus fertilis (Steud.) Clayton, were recorded as C-4 plants for the first time. The biochemical subdivisions of these C-4 weeds were also determined. As in the natural C-4 populations, the NADP-malic enzyme subtype of C-4 photosynthesis dominates the list of C-4 weeds on this island (62%), while the PEP carboxykinase subtype is relatively rare (12%). NAD-malic enzyme subtype has an intermediate representation (26%). The high proportion of weeds in Taiwan being C-3 plants is noteworthy, and it may be accounted for by the high precipitation in this subtropical island

The promoter for the maize C-4 pyruvate,orthophosphate dikinase gene directs cell- and tissue-specific transcription in transgenic maize plants

The pyruvate, orthophosphate dikinase (PPDK) gene coding the chloroplast enzyme involved in C-4 photosynthesis has a dual promoter system, The first promoter is responsible for the transcription of a larger transcript and its product is targeted to the chloroplast (hence, it is designated as C4Pdk promoter) while the second promoter is responsible for the transcription of a smaller transcript and its product remains in the cytosol. In this study, chimeric maize C4Pdk promoter (0.9 or 1.5 kb)-beta-glucuronidase or luciferase fusion genes were introduced into maize plants by Agrobacterium-mediated transformation. The cell- and tissue-specificities of the maize C4Pdk promoter in the transgenic maize plants were examined by histochemical and enzymic activity analyses of the reporters in different photosynthetic cells and tissues. The results showed that the reporter proteins are almost exclusively localized in leaf mesophyll cells, Among the tissues tested, leaf blade had the highest reporter activities with sheath exhibiting about 10% of the activities in blade. Husk, stem, tassel and root had no or very little reporter activities. Taken together, these results suggest that the maize C4Pdk promoter is specifically transcribed in the mesophyll cells of leaf blade and to a much less extent in the mesophyll cells of sheath, but not in leaf bundle sheath cells or other tissues. Furthermore, the 0.9 kb maize C4Pdk promoter sequences appear to contain the necessary cis-acting elements for its cell- and organ-specific expression

The evolution of C4 plants: acquisition of cis-regulatory sequences in the promoter of C4-type pyruvate, orthophosphate dikinase gene

In a previous study, we identified the C4-like pyruvate, orthophosphate dikinase gene (Pdk) in the C3 plant rice, with a similar structure to the C4-type Pdk in the C4 plant maize. In order to elucidate the differences between C4-type and C4-like Pdk genes in C4 and C3 plants, we have produced chimeric constructs with the P-glucuronidase (GUS) reporter gene under the control of the Pdk promoters. In transgenic rice, both rice and maize promoters directed GUS expression in photosynthetic organs in a light-dependent manner. However, the maize promoter exhibited a much higher transcriptional activity than the rice promoter did. These results indicate that the rice C4-like Pdk gene resembles the maize C4-type Pdk gene in terms of regulation of expression. We also tested the activity of the rice promoter in transgenic maize. GUS activity was seen in both photosynthetic and non-photosynthetic organs. Thus, the rice promoter does not confer a strict organ-specific gene expression, as the maize promoter does. Moreover, the rice promoter directed GUS expression not only in mesophyll cells but also in bundle sheath cells, whereas the maize promoter directed expression only in mesophyll cells. Taken together, the results obtained from both transgenic maize and rice demonstrate that the rice and maize promoters differ not only quantitatively, but also qualitatively, in terms of their cell- and organ-specificity. Experiments with swapped promoters using the rice and maize promoters further demonstrated that a limited sequence region from -330 to -76 of the maize promoter confers light-regulated, high-level expression to the rice promoter in maize mesophyll protoplasts. We conclude the gain of cis-acting elements conferring high-level expression and mesophyll cell specificity was necessary for establishment of a C4-type Pdk gene during the course of evolution from C3 to C4 plants