NAD-malic Enzyme-type C4 Plants Research Articles

Aggregative movement of C4 mesophyll chloroplasts is promoted by low CO2 under high intensity blue light.

C4 plants supply concentrated CO2 to bundle sheath (BS) cells, improving photosynthetic efficiency by suppressing photorespiration. Mesophyll chloroplasts in C4 plants are redistributed toward the sides of the BS cells (aggregative movement) in response to environmental stresses under light. Although this chloroplast movement is common in C4 plants, the significance and mechanisms underlying the aggregative movement remain unknown. Under environmental stresses, such as drought and salt, CO2 uptake from the atmosphere is suppressed by closing stomata to prevent water loss. We hypothesized that CO2 limitation may induce the chloroplast aggregative movement. In this study, the mesophyll chloroplast arrangement in a leaf of finger millet, an NAD-malic enzyme type C4 plant, was examined under different CO2 concentrations and light conditions. CO2 limitation around the leaves promoted the aggregative movement, but the aggregative movement was not suppressed, even at the higher CO2 concentration than in the atmosphere, under high intensity blue light. In addition, mesophyll chloroplasts did not change their arrangement under darkness or red light. From these results, it can be concluded that CO2 limitation is not a direct inducer of the aggregative movement but would be a promoting factor of the movement under high intensity blue light.

Interaction of polyethylene glycol-6000 with C4 phosphoenolpyruvate carboxylase in crude leaf extracts as well as in purified protein form from Amaranthus hypochondriacus L.: evidence for oligomerization of PEPC in vitro and in vivo

The interaction of phosphoenolpyruvate carboxylase (PEPC) with a compatible solute, PEG-6000, was examined using crude leaf extracts as well as the purified protein from leaves of Amaranthus hypochondriacus, a NAD-malic enzyme type C4 plant. The inclusion in the assay medium of PEG-6000 stimulated the activity of purified PEPC by about 2.5-fold over control. The addition of PEG during both extraction and assay, stimulated PEPC activity by almost 5.0 fold in crude extracts. The stimulation by PEG of the dark-form of PEPC (2.4 fold) was more than that of the light-form (1.7 fold). Gel filtration of PEPC in leaf extracts on Sephadex G-200, showed the existence of three different oligomeric forms: tetramer, dimer and monomer. The exclusion of PEG and glycerol during extraction and elution on Sephadex resulted in a marked shift of the enzyme into dimer and/or monomer, with a very small proportion of tetramer but on the contrary, the inclusion of PEG and glycerol resulted in the enzyme maintaining predominantly a tetrameric shape. Thus, the activity and the structural properties of PEPC can be influenced by the presence or absence of compatible solutes (PEG or glycerol), obviously due to changes in the microenvironment of the enzyme.

The Expression of 2-Oxoglutarate/Malate Translocator in the Bundle-Sheath Mitochondria of Panicum miliaceum, a NAD-Malic Enzyme-Type C4 Plant, Is Regulated by Light and Development.

The bundle-sheath mitochondria in NAD-malic enzyme-type C4 plants participate in the C4 dicarboxylate cycle and require high capacities of translocators to accommodate the high rates of exchange of metabolites involved in photosynthesis. In Panicum miliaceum, a NAD-malic enzyme-type C4 plant, the steady-state level of mRNA for the mitochondrial 2-oxoglutarate (2-OG)/malate translocator was higher in leaves than in nonphotosynthetic tissues. Furthermore, the expression of the gene for the mitochondrial 2-OG/malate translocator was restricted to bundle-sheath cells (BSC) but not mesophyll cells. The transcript level of the BSC-located mitochondrial 2-OG/malate translocator increased during greening in accordance with levels of photosynthetic genes, although the relative transcript levels of other mitochondrial membrane proteins decreased. The specific activities of C4 photosynthetic enzymes and the relative abundance of the 2-OG/malate translocator protein in bundle-sheath mitochondria increased in successive sections from the basal meristem to the distal tip, whereas the specific activities of mitochondrial respiratory enzymes remained constant or decreased. These findings indicate that the specific 2-OG/malate translocator in BSC mitochondria of P. miliaceum is expressed in concert with C4 enzymes during the differentiation of BSC and parallels the capacity of C4 photosynthesis. Most unusual, northern analysis showed that significant amounts of unspliced mRNAs, the levels of which are variable during greening, were present in leaf tissues. It is possible that this incomplete splicing is involved in posttranscriptional regulation of expression of this gene.

Phosphoenolpyruvate Carboxykinase in C4 Plants: Its Role and Regulation

Some of the recent findings which revise our view of the role and regulation of phosphoenolpyruvate carboxykinase (PEPCK) in C4 plants are discussed. Evidence is presented that PEPCK is present at appreciable activities in the bundle-sheath of some NADP-malic enzyme-type C4 plants, such as maize, but it was not detectable in NAD-malic enzyme-type C4 plants. PEPCK is rapidly inactivated in crude extracts of leaves of the C4 plant, Panicum maximum. This inactivation could be prevented by high concentrations of dithiothreitol or by the inclusion of ADP or ATP, suggesting the involvement of thiols at the active site. PEPCK is also subject to rapid proteolysis in crude extracts of a range of C4 plants, resulting in cleavage to a smaller (62 kDa) form. This can be reduced by extraction at high pH and by the inclusion of SDS, but it means that intact PEPCK has never been purified from a C4 plant. The molecular mass of PEPCK varies considerably in C4 plants, unlike C3 and CAM plants in which it is usually 74 kDa. PEPCK is phosphorylated during darkness (and reversed by light) in some C4 plants with PEPCK of a larger molecular mass, such as Panicum maximum (71 kDa), but it was not phosphorylated in the PEPCK-type C4 plant, Sporobolus pyramidalis (69 kDa). The known regulatory properties of PEPCK are discussed in relation to its role in C4 photosynthesis, in particular its sensitivity to regulation by adenylates and by Mn2+.

Isolation, characterization and expression of cDNA clones encoding a mitochondrial malate translocator from Panicum miliaceum L.

Three cDNA clones that hybridize to a partial rice cDNA that show similarity to bovine mitochondrial 2-oxoglutarate/malate translocator were isolated from leaves of Panicum miliaceum L. (proso millet), an NAD-malic enzyme-type C4 plant. The nucleotide sequences of the clones resemble each other, and some of the isolated cDNAs contained extra sequences that seemed to be introns. The predicted proteins encoded by the cDNAs have 302 amino acids and molecular weights of 32211 and 32150. The hydrophobic profile of the amino acid sequence predicted the existence of six transmembrane alpha-helices that is a common property of members in the mitochondrial transporter family. The predicted amino acid sequence showed the highest similarity with that of the 2-oxoglutarate/malate translocator from mammalian mitochondria. An expression plasmid containing the coding region of the cDNAs was used to over-express recombinant protein with a C-terminal histidine tag Escherichia coli, which was affinity purified. The antibody against the recombinant protein cross-reacted with proteins of 31-32 kDa in the membrane fraction from P. miliaceum mitochondria, but not with the chloroplast fraction. The recombinant protein reconstituted in liposomes efficiently transported malate, citrate, and 2-oxoglutarate.

Molecular cloning of an alanine aminotransferase from NAD-malic enzyme type C4 plant Panicum miliaceum.

We have determined the nucleotide sequence of a cDNA encoding AlaAT-2, which is believed to function in the C4-pathway of Panicum miliaceum. An open reading frame (1446 bp) encodes a protein of 482 amino acid residues. The deduced amino acid sequence of AlaAT-2 shows 44.2 and 44.8% homology with the amino acid sequences of AlaATs from rat and human livers, respectively. Northern blot analysis showed that the gene encoding AlaAT-2 in mesophyll and bundle sheath cells was the same and transcribed similarly in the cells. The level of translatable mRNA for AlaAT-2 increased dramatically during greening.