NADP-malic Enzyme-type C4 Plants Research Articles

A two-dimensional microscale model of gas exchange during photosynthesis in maize (Zea mays L.) leaves

CO2 exchange in leaves of maize (Zea mays L.) was examined using a microscale model of combined gas diffusion and C4 photosynthesis kinetics at the leaf tissue level. Based on a generalized scheme of photosynthesis in NADP-malic enzyme type C4 plants, the model accounted for CO2 diffusion in a leaf tissue, CO2 hydration and assimilation in mesophyll cells, CO2 release from decarboxylation of C4 acids, CO2 fixation in bundle sheath cells and CO2 retro-diffusion from bundle sheath cells. The transport equations were solved over a realistic 2-D geometry of the Kranz anatomy obtained from light microscopy images. The predicted responses of photosynthesis rate to changes in ambient CO2 and irradiance compared well with those obtained from gas exchange measurements. A sensitivity analysis showed that the CO2 permeability of the mesophyll-bundle sheath and airspace–mesophyll interfaces strongly affected the rate of photosynthesis and bundle sheath conductance. Carbonic anhydrase influenced the rate of photosynthesis, especially at low intercellular CO2 levels. In addition, the suberin layer at the exposed surface of the bundle sheath cells was found beneficial in reducing the retro-diffusion. The model may serve as a tool to investigate CO2 diffusion further in relation to the Kranz anatomy in C4 plants.

Light-regulated phosphorylation of maize phosphoenolpyruvate carboxykinase plays a vital role in its activity

Phosphoenolpyruvate carboxykinase (PEPCK)-the major decarboxylase in PEPCK-type C4 plants-is also present in appreciable amounts in the bundle sheath cells of NADP-malic enzyme-type C4 plants, such as maize (Zea mays), where it plays an apparent crucial role during photosynthesis (Wingler et al., in Plant Physiol 120(2):539-546, 1999; Furumoto et al., in Plant Mol Biol 41(3):301-311, 1999). Herein, we describe the use of mass spectrometry to demonstrate phosphorylation of maize PEPCK residues Ser55, Thr58, Thr59, and Thr120. Western blotting indicated that the extent of Ser55 phosphorylation dramatically increases in the leaves of maize seedlings when the seedlings are transferred from darkness to light, and decreases in the leaves of seedlings transferred from light to darkness. The effect of light on phosphorylation of this residue is opposite that of the effect of light on PEPCK activity, with the decarboxylase activity of PEPCK being less in illuminated leaves than in leaves left in the dark. This inverse relationship between PEPCK activity and the extent of phosphorylation suggests that the suppressive effect of light on PEPCK decarboxylation activity might be mediated by reversible phosphorylation of Ser55.

Consequences of C4 Differentiation for Chloroplast Membrane Proteomes in Maize Mesophyll and Bundle Sheath Cells

Chloroplasts of maize leaves differentiate into specific bundle sheath (BS) and mesophyll (M) types to accommodate C(4) photosynthesis. Chloroplasts contain thylakoid and envelope membranes that contain the photosynthetic machineries and transporters but also proteins involved in e.g. protein homeostasis. These chloroplast membranes must be specialized within each cell type to accommodate C(4) photosynthesis and regulate metabolic fluxes and activities. This quantitative study determined the differentiated state of BS and M chloroplast thylakoid and envelope membrane proteomes and their oligomeric states using innovative gel-based and mass spectrometry-based protein quantifications. This included native gels, iTRAQ, and label-free quantification using an LTQ-Orbitrap. Subunits of Photosystems I and II, the cytochrome b(6)f, and ATP synthase complexes showed average BS/M accumulation ratios of 1.6, 0.45, 1.0, and 1.33, respectively, whereas ratios for the light-harvesting complex I and II families were 1.72 and 0.68, respectively. A 1000-kDa BS-specific NAD(P)H dehydrogenase complex with associated proteins of unknown function containing more than 15 proteins was observed; we speculate that this novel complex possibly functions in inorganic carbon concentration when carboxylation rates by ribulose-bisphosphate carboxylase/oxygenase are lower than decarboxylation rates by malic enzyme. Differential accumulation of thylakoid proteases (Egy and DegP), state transition kinases (STN7,8), and Photosystem I and II assembly factors was observed, suggesting that cell-specific photosynthetic electron transport depends on post-translational regulatory mechanisms. BS/M ratios for inner envelope transporters phosphoenolpyruvate/P(i) translocator, Dit1, Dit2, and Mex1 were determined and reflect metabolic fluxes in carbon metabolism. A wide variety of hundreds of other proteins showed differential BS/M accumulation. Mass spectral information and functional annotations are available through the Plant Proteome Database. These data are integrated with previous data, resulting in a model for C(4) photosynthesis, thereby providing new rationales for metabolic engineering of C(4) pathways and targeted analysis of genetic networks that coordinate C(4) differentiation.

High level expression of C4-specific NADP-malic enzyme in leaves and impairment of photoautotrophic growth in a C3 plant, rice.

The chloroplastic NADP-malic enzyme (NADP-ME) is a key enzyme of the C4 photosynthesis pathway in NADP-ME type C4 plants such as maize. To express the chloroplastic NADP-ME in leaves of a C3 plant, rice, full-length cDNAs encoding the rice C3-specific isoform and the maize C4-specific isoform of the enzyme were expressed under the control of the rice CAB: promoter. Transformants carrying the rice cDNA showed the NADP-ME activities in the leaves less than several-fold that of non-transformants, while those carrying the maize cDNA showed activities up to 30-fold that of non-transformants or about 60% of the NADP-ME activity of maize leaves. These results indicate that expression of the rice C3-specific NADP-ME is suppressed at co- and/or post-transcriptional levels by some regulation mechanisms intrinsic to rice, while that of the foreign C4-specific isoform can escape from such suppression. The accumulation of the maize C4-specific NADP-ME led to bleaching of leaf color and growth hindrance in rice plants under natural light. These deteriorative effects resulted from enhanced photoinhibition of photosynthesis due to an increase in the level of NADPH inside the chloroplast by the action of the maize enzyme.

A nuclear-encoded protein of prokaryotic origin is essential for the stability of photosystem II in Arabidopsis thaliana.

To understand the regulatory mechanisms underlying the biogenesis of photosystem II (PSII) we have characterized the nuclear mutant hcf136 of Arabidopsis thaliana and isolated the affected gene. The mutant is devoid of any photosystem II activity, and none of the nuclear- and plastome-encoded subunits of this photosystem accumulate to significant levels. Protein labelling studies in the presence of cycloheximide showed that the plastome-encoded PSII subunits are synthesized but are not stable. The HCF136 gene was isolated by virtue of its T-DNA tag, and its identity was confirmed by complementation of homozygous hcf136 seedlings. Immunoblot analysis of fractionated chloroplasts showed that the HCF136 protein is a lumenal protein, found only in stromal thylakoid lamellae. The HCF136 protein is produced already in dark-grown seedlings and its levels do not increase dramatically during light-induced greening. This accumulation profile confirms the mutational data by showing that the HCF136 protein must be present when PSII complexes are made. HCF136 homologues are found in the cyanobacterium Synechocystis species PCC6803 (slr2034) and the cyanelle genome of Cyanophora paradoxa (ORF333), but are lacking in the plastomes of chlorophytes and metaphytes as well as from those of rhodo- and chromophytes. We conclude that HCF136 encodes a stability and/or assembly factor of PSII which dates back to the cyanobacterial-like endosymbiont that led to the plastids of the present photosynthetic eukaryotes.

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.

Differential biogenesis of photosystem II in mesophyll and bundle-sheath cells of monocotyledonous NADP-malic enzyme-type C4 plants: the non-stoichiometric abundance of the subunits of photosystem II in the bundle-sheath chloroplasts and the translational activity of the plastome-encoded genes

Photosystem II activities as well as polypeptide levels were compared in mesophyll and bundle-sheath chloroplasts of the two monocotyledonous NADP-malic enzyme-type C4 species Zea may L. (maize) and Sorghum bicolor (L.) Moench. Small decreases in PSII activities as well as protein levels were observed when comparing maize seedlings of varying ages. In contrast, PSII activity was not noticeable in the bundle-sheath chloroplasts of even very young Sorghum seedlings, whereas substantial amounts of some of the PSII polypeptides analyzed were still detectable. In both species the degree of reduction was found to vary drastically with respect to the different PSII subunits. The greatest depletion was observed for the 23- and 16-kDa regulatory polypeptides of the water-splitting apparatus and the 10-kDa polypeptide (psbR product), indicating that PSII activity in bundle-sheath chloroplasts is limited due to a depletion at the oxidizing site of this complex. To gain insight into the regulatory principles which control the cell-specific expression of the plastome-encoded PSII genes an in-organello translation system with intact mesophyll and bundle-sheath chloroplasts of maize was established. The synthesis of the D1 polypeptide of the PSII reaction center was found to be reduced in bundle-sheath chloroplasts, reflecting the de creased levels of Dl in this type of chloroplast.

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.

Hill Reaction, Hydrogen Peroxide Scavenging, and Ascorbate Peroxidase Activity of Mesophyll and Bundle Sheath Chloroplasts of NADP-Malic Enzyme Type C4 Species

Intact mesophyll and bundle sheath chloroplasts wee isolated from the NADP-malic enzyme type C(4) plants maize, sorghum (monocots), and Flaveria trinervia (dicot) using enzymic digestion and mechanical isolation techniques. Bundle sheath chloroplasts of this C(4) subgroup tend to be agranal and were previously reported to be deficient in photosystem II activity. However, following injection of intact bundle sheath chloroplasts into hypotonic medium, thylakoids had high Hill reaction activity, similar to that of mesophyll chloroplasts with the Hill oxidants dichlorophenolindophenol, p-benzoquinone, and ferricyanide (approximately 200 to 300 micromoles O(2) evolved per mg chlorophyll per hour). In comparison to that of mesophyll chloroplasts, the Hill reaction activity of bundle sheath chloroplasts of maize and sorghum was labile and lost activity during assay. Bundle sheath chloroplasts of maize also exhibited some capacity for 3-phosphoglycerate dependent O(2) evolution (29 to 58 micromoles O(2) evolved per milligram chlorophyll per hour). Both the mesophyll and bundle sheath chloroplasts were equally effective in light dependent scavenging of hydrogen peroxide. The results suggest that both chloroplast types have noncyclic electron transport and the enzymology to reduce hydrogen peroxide to water. The activities of ascorbate peroxidase from these chloroplast types was consistent with their capacity to scavenge hydrogen peroxide.

Bundle-sheath thylakoids from NADP-malic enzyme-type C4 plants require an exogenous electron donor for enzyme light activation

Light activation of either NADP-malate dehydrogenase (EC 1.1.1.82) or fructose-1,6-bisphosphate phosphatase (EC 3.1.3.11) was assayed in a reconstituted chloroplastic, system comprising the isolated proteins of the ferredoxin-thioredoxin light-activation system and thylakoids from either mesophyll or bundle-sheath tissues of different C4 plants. While C4-plant thylakoids functionned almost equally well with C3-or C4-plant proteins, the photosyntem-II-deficient bundle-sheath thylakoids from the NADP-malic enzyme type, were unable to perform enzyme photoactivation unless supplemented with an electron donor to photosystem I. Bundle-sheath thylakoids isolated from plants showing no photosystem-II deficiency did not require such an addition. The results are discussed with respect to a possible requirement for a physiological reductant of ferredoxin for enzyme light activation in bundle-sheath, tissues.