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1.
Activity regulation and physiological impacts of maize C4-specific phosphoenolpyruvate carboxylase overproduced in transgenic rice plants 总被引:6,自引:0,他引:6
Fukayama H Hatch MD Tamai T Tsuchida H Sudoh S Furbank RT Miyao M 《Photosynthesis research》2003,77(2-3):227-239
Phosphoenolpyruvate carboxylase (PEPC) was overproduced in the leaves of rice plants by introducing the intact maize C4-specific PEPC gene. Maize PEPC in transgenic rice leaves underwent activity regulation through protein phosphorylation in
a manner similar to endogenous rice PEPC but contrary to that occurring in maize leaves, being downregulated in the light
and upregulated in the dark. Compared with untransformed rice, the level of the substrate for PEPC (phosphoenolpyruvate) was slightly lower and the product (oxaloacetate) was slightly higher in transgenic rice, suggesting that maize
PEPC was functioning even though it remained dephosphorylated and less active in the light. 14CO2 labeling experiments indicated that maize PEPC did not contribute significantly to the photosynthetic CO2 fixation of transgenic rice plants. Rather, it slightly lowered the CO2 assimilation rate. This effect was ascribable to the stimulation of respiration in the light, which was more marked at lower
O2 concentrations. It was concluded that overproduction of PEPC does not directly affect photosynthesis significantly but it
suppresses photosynthesis indirectly by stimulating respiration in the light. We also found that while the steady-state stomatal
aperture remained unaffected over a wide range of humidity, the stomatal opening under non-steady-state conditions was destabilized
in transgenic rice.
This revised version was published online in August 2006 with corrections to the Cover Date. 相似文献
2.
Outlaw WH Du Z Xia Meng F Aghoram K Riddle KA Chollet R 《Archives of biochemistry and biophysics》2002,407(1):63-71
Leaves regulate gas exchange through control of stomata in the epidermis. Stomatal aperture increases when the flanking guard cells accumulate K+ or other osmolytes. K+ accumulation is stoichiometric with H+ extrusion, which is compensated for by phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31)-mediated malate synthesis. Plant PEPCs are regulated allosterically and by phosphorylation. Aspects of the signal-transduction network that control the PEPC phosphorylation state in guard cells are reported here. Guard cells were preloaded with [32P]orthophosphate (32Pi); then stomata were incubated with fusicoccin (FC), which activates the guard-cell plasma membrane H+-ATPase. [32P]PEPC was assessed by immunoprecipitation, electrophoresis, immunoblotting, and autoradiography. In -FC controls, stomatal size, guard-cell malate, and [32P]PEPC were low; maximum values for these parameters were observed in the presence of FC after a 90-min incubation and persisted for an additional 90 min. This high steady-state phosphorylation status resulted from continuous phosphorylation and dephosphorylation, even after the malate-accumulation phase. PEPC phosphorylation was diminished by approximately 80% when K+ uptake was associated with Cl- uptake and was essentially abolished when stomatal opening was sucrose--rather than K+--dependent. Finally, alkalinization by NH4+ in the presence of K+ did not cause PEPC phosphorylation (as it does in C4 plants). As discussed, a role for cytoplasmic protons cannot be completely excluded by this result. In summary, activation of the plasma membrane H+-ATPase was essential, but not sufficient, to cause phosphorylation of guard-cell PEPC. Network components downstream of the H+-ATPase influence the phosphorylation state of this PEPC isoform. 相似文献
3.
Phosphoenolpyruvate carboxylase (PEPCase, EC 4.1.1.3) is a key enzyme of C4 photosynthesis. It has evolved from ancestral non-photosynthetic (C3) isoforms and thereby changed its kinetic and regulatory properties. We are interested in understanding the molecular changes,
as the C4 PEPCases were adapted to their new function in C4 photosynthesis and have therefore analysed the PEPCase genes of various Alternanthera species. We isolated PEPCase cDNAs from the C4 plant Alternanthera pungens H.B.K., the C3/C4 intermediate plant A. tenella Colla, and the C3 plant A. sessilis (L.) R.Br. and investigated the kinetic properties of the corresponding recombinant PEPCase proteins and their phylogenetic
relationships. The three PEPCases are most likely derived from orthologous gene classes named ppcA. The affinity constant for the substrate phosphoenolpyruvate (K
0.5 PEP) and the degree of activation by glucose-6-phosphate classified the enzyme from A. pungens (C4) as a C4 PEPCase isoform. In contrast, both the PEPCases from A. sessilis (C3) and A. tenella (C3/C4) were found to be typical C3 PEPCase isozymes. The C4 characteristics of the PEPCase of A. pungens were accompanied by the presence of the C4-invariant serine residue at position 775 reinforcing that a serine at this position is essential for being a C4 PEPCase (Svensson et al. 2003). Genomic Southern blot experiments and sequence analysis of the 3′ untranslated regions of these genes indicated the existence
of PEPCase multigene family in all three plants which can be grouped into three classes named ppcA, ppcB and ppcC. 相似文献
4.
In order to elucidate the discrete steps in phospho enolpyruvate carboxylase (PEPC) evolution concerning K(m)-PEP and malate tolerance a comparison was made between C3, C3-C4 and C4 species of the dicot genus Flaveria. The PEPCs of this genus are encoded by a gene family comprising three classes: ppcA, ppcB and ppcC [J. Hermans and P. Westhoff (1990) Mol Gen Genet 224:459-468, (1992) Mol Gen Genet 234:275-284]. The ppcA of F trinervia (C4) codes for the C4 PEPC isoform but other plants of the genus contain ppcA orthologues too. The C3 plant F. pringlei showed the lowest levels of ppcA PEPC mRNA followed by F. pubescens (C3-C4) while the C4-like plant F. brownii displayed RNA amounts close to the C4 species F. trinervia. In contrast to the similar expression profiles of F. brownii (C4-like) and F. trinervia (C4) the PEPC amino acid sequence of F. brownii was more similar to the C3 and C3-C4 ppcA PEPCs than to the C4 PEPC. Similarly, the C3, C3-C4 and C4-like ppcA PEPCs showed almost identical PEP saturation kinetics when activated by glucose-6-phosphate ( K(m)-PEP: 17-20 microM) while the K(m)-PEP for the C4 PEPC was determined to be 53 microM. However, without activation the ppcA PEPCs of F. pubescens and F. brownii displayed C3-C4 intermediate values. A similar picture was obtained when the malate sensitivities were compared. In the non-activated state the F. trinervia (C4) enzyme was 10 times more tolerant to malate than the F. pringlei counterpart. The ppcA enzymes of F. pubescens (C3-C4) and F. brownii (C4-like) displayed intermediate values. In contrast, the inclusion of 5 mM glucose-6-phosphate in the reaction mixture changed the order totally. Interestingly, the activation rendered the C4 enzyme about 50% less tolerant to malate than the C3 PEPC. The activation had a positive effect on malate tolerance of the F. pubescens (C3-C4) PEPC while the ppcA PEPC of F. brownii (C4-like) was almost unaffected. 相似文献
5.
Characterization of a novel class of plant homeodomain proteins that bind to the C4 phosphoenolpyruvate carboxylase gene of Flaveria trinervia 总被引:1,自引:0,他引:1
We are interested in the regulatory mechanisms responsible for the mesophyll-specific expression of C4 phosphoenolpyruvate carboxylase (PEPCase). A one-hybrid screen resulted in the cloning of four different members of a novel class of plant homeodomain proteins, which are most likely involved in the mesophyll-specific expression of the C4 PEPCase gene in C4 species of the genus Flaveria. Inspection of the homeodomains of the four proteins reveals that they share many common features with homeodomains described so far, but there are also significant differences. Interestingly, this class of homeodomain proteins occurs also in Arabidopsis thaliana and other C3 plants. One-hybrid experiments as well as in vitro DNA binding studies confirmed that these novel homeodomain proteins specifically interact with the proximal region of the C4 PEPCase gene. The N-terminal domains of the homeodomain proteins contain highly conserved sequence motifs. Two-hybrid experiments show that these motifs are sufficient to confer homo- or heterodimer formation between the proteins. Mutagenesis of conserved cysteine residues within the dimerization domain indicates that these residues are essential for dimer formation. Therefore, we designate this novel class of homeobox proteins ZF-HD, for zinc finger homeodomain protein. Our data suggest that the ZF-HD class of homeodomain proteins may be involved in the establishment of the characteristic expression pattern of the C4 PEPCase gene. 相似文献
6.
A rabbit antiserum was raised against phosphoenolpyruvate carboxykinase (PCK) purified from Urochloa panicoides, a PCK-type C4 monocot. The antiserum was used to screen a cDNA expression library constructed from U. panicoides leaf poly(A)+RNA. Inserts from immunoreactive clones were used to rescreen the library and obtain three overlapping cDNAs comprising a 2220 bp composite sequence. The single complete open reading frame of 1872 bp encodes PCK1, a 624 amino acid polypeptide with a predicted molecular mass of 68474 Da. Comparison of PCK1 with other ATP-dependent PCKs indicates that PCK1 is significantly larger, mainly due to an N-terminal extension of greater than 65 residues, and reveals high sequence identity across the central portion of the protein, especially over seven sub-sequences. One of these sub-sequences spans motifs common to several ATP-utilising enzymes for phosphate and divalent cation binding. The anti-PCK antiserum recognises a 69 kDa polypeptide on immunoblots of either purified PCK or U. panicoides leaf extracts. However, polypeptides of 63, 62, 61 and 60 kDa are also immunoreactive. Amino terminal sequencing of polypeptides from preparations of purified PCK demonstrates that these smaller polypeptides are related to PCK1, and time course experiments show that these polypeptides arise from the breakdown of PCK during isolation. Northern blot analysis indicates that the 2.7 kb PCK mRNA is abundant in green leaves but not in roots or etiolated shoots. Moreover, PCK mRNA levels increase gradually during greening, reaching maximum levels after about 84 h. 相似文献
7.
Phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) protein kinase (PPCK) was purified ∼1,500-fold from developing castor oil seeds (COS).
Gel filtration and immunoblotting with anti-(rice PPCK2)-immune serum indicated that this Ca2+-insensitive PPCK exists as a 31-kDa monomer. COS PPCK-mediated rephosphorylation of the 107-kDa subunit (p107) of COS PEPC1
(K
m = 2.2 μM) activated PEPC1 by ∼80% when assayed under suboptimal conditions (pH 7.3, 0.2 mM PEP, and 0.125 mM malate). COS
PPCK displayed remarkable selectivity for phosphorylating COS PEPC1 (relative to tobacco, sorghum, or maize PEPCs), exhibited
a broad pH-activity optima of ∼pH 8.5, and at pH 7.3 was activated 40–65% by 1 mM PEP, or 10 mM Gln or Asn, but inhibited
65% by 10 mM L-malate. The possible control of COS PPCK by disulfide-dithiol interconversion was suggested by its rapid inactivation
and subsequent reactivation when incubated with oxidized glutathione and then dithiothreitol. In vitro PPCK activity correlated
with in vivo p107 phosphorylation status, with both peaking in mid-cotyledon to full-cotyledon developing COS. Notably, PPCK
activity and p107 phosphorylation of developing COS were eliminated following pod excision or prolonged darkness of intact
plants. Both effects were fully reversed 12 h following reillumination of darkened plants. These results implicate a direct
relationship between the up-regulation of COS PPCK and p107 phosphorylation during the recommencement of photosynthate delivery
from illuminated leaves to the non-photosynthetic COS. Overall, the results support the hypothesis that PEPC and PPCK participate
in the control of photosynthate partitioning into C-skeletons needed as precursors for key biosynthetic pathways of developing
COS.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
8.
The occurrence of both C3 and C4 photosynthetic characteristics in a single Zea mays plant 总被引:1,自引:0,他引:1
The activities of the carboxylating enzymes ribulose-1,5-biphosphate (RuBP) carboxylase and phosphoenolpyruvate (PEP) carboxylase in leaves of three-week old Zea mays plants grown under phytotron conditions were found to vary according to leaf position. In the lower leaves the activity of PEP carboxylase was lower than that of RuBP carboxylase, while the upper leaves exhibited high levels of PEP carboxylase. Carbon dioxide compensation points and net photosynthetic rates also differed in the lower and upper leaves. Differences in the fine structure of the lowermost and uppermost leaves are shown. The existence of both the C3 and C4 photosynthetic pathways in the same plant, in this and other species, is discussed.Abbreviations PEP
phosphoenolpyruvate
- RuBP
ribulose-1,5-biphosphate 相似文献
9.
In C4 grasses belonging to the NADP-malic enzyme-type subgroup, malate is considered to be the predominant C4 acid metabolized during C4 photosynthesis, and the bundle sheath cell chloroplasts contain very little photosystem-II (PSII) activity. The present studies showed that Flaveria bidentis (L.), an NADP-malic enzyme-type C4 dicotyledon, had substantial PSII activity in bundle sheath cells and that malate and aspartate apparently contributed about equally to the transfer of CO2 to bundle sheath cells. Preparations of bundle sheath cells and chloroplasts isolated from these cells evolved O2 at rates between 1.5 and 2 mol · min–1 · mg–1 chlorophyll (Chl) in the light in response to adding either 3-phosphoglycerate plus HCO
3
–
or aspartate plus 2-oxoglutarate. Rates of more than 2 mol O2 · min–1 · mg–1 Chl were recorded for cells provided with both sets of these substrates. With bundle sheath cell preparations the maximum rates of light-dependent CO2 fixation and malate decarboxylation to pyruvate recorded were about 1.7 mol · min–1 · mg–1 Chl. Compared with NADP-malic enzyme-type grass species, F. bidentis bundle sheath cells contained much higher activities of NADP-malate dehydrogenase and of aspartate and alanine aminotransferases. Time-course and pulse-chase studies following the kinetics of radiolabelling of the C-4 carboxyl of C4 acids from 14CO2 indicated that the photosynthetically active pool of malate was about twice the size of the aspartate pool. However, there was strong evidence for a rapid flux of carbon through both these pools. Possible routes of aspartate metabolism and the relationship between this metabolism and PSII activity in bundle sheath cells are considered.Abbreviations DHAP
dihydroxyacetone phosphate
- NADP-ME(-type)
NADP-malic enzyme (type)
- NADP-MDH
NADP-malate dehydrogenase
- OAA
oxaloacetic acid
- 2-OG
2-oxoglutarate
- PEP
phosphoenolpyruvate
- PGA
3-phosphoglycerate
- Pi
orthophosphate
- Ru5P
ribulose 5-phosphate 相似文献
10.
Isolated mesophyll cells from darkened leaves of the C(4) plant Digitaria sanguinalis keep functional plasmodesmata that allow the free exchange of low molecular mass compounds with the surrounding medium. This cell suspension system has been used to measure C(4) PEPC activity in situ using a spectrophotometric assay. Compared to the extracted enzyme assayed in vitro, the essentially non-phosphorylated 'in-cell' C(4) PEPC showed altered functional and regulatory properties. While the S (0.5) for PEP at pH 7.3 was only modestly changed (0.4-0.6 mM), the response to pH was shifted towards the acidic range, being close to the maximal value at pH 7.3. Using expected physiological concentrations of the metabolites, at pH 7.3, the IC(50) for malate showed a five-fold increase, from 1.5 to 8 mM, and was increased further to 22 mM in the presence of the allosteric activator glucose-6-phosphate (4 mM). Thiol compounds like DTT, mercaptoethanol and reduced glutathione weakened the in-situ sensitivity of C(4) PEPC to malate. However, none of them had any effect on this process in vitro. This was not due to thioredoxin-mediated or phoshorylation-dependent processes. Since glutathione is a physiological compound that is present mostly in the reduced state in the cell cytosol, a possible contribution of this thiol to the protection of the enzyme against malate in situ is proposed. 相似文献
11.
Osamu Ueno 《Planta》1996,199(3):382-393
Eleocharis vivipara Link is a unique amphibious leafless sedge. The terrestrial form has Kranz anatomy and the biochemical traits of C4 plants while the submerged form develops structural and biochemical traits similar to those of C3 plants. The structural features of the culms, which are the photosynthetic organs, of the two forms were examined and compared. The culms of the terrestrial form have mesophyll cells and three bundle sheaths which consist of three kinds of cell, namely, the innermost Kranz cells that contain large numbers of organelles, the middle mestome sheath cells that lack chloroplasts, and the outermost parenchyma sheath cells that contain chloroplasts. The culms of the submerged form had a tendency towards reduction in numbers and size of Kranz cells and vascular bundles, as compared to the terrestrial form, and they had spherical mesophyll cells that were tightly packed without intercellular spaces inside the epidermis. The submerged form had a higher ratio of cross-sectional area of mesophyll cells plus parenchyma sheath cells to that of Kranz cells than the terrestrial form. The difference was mainly due to a decrease in the number and the size of the Kranz cells and to a marked increase in the size of the mesophyll cells and the parenchyma sheath cells in the submerged form, as compared to the terrestrial form. The Kranz cells of the terrestrial form had basically the structural characteristics of plants of the NAD-malic enzyme type, with the exception of the intracellular location of organelles. The Kranz cells of the submerged form included only a few organelles, and the percentage of organelles partitioned to the Kranz cells was significantly smaller in the submerged form than in the terrestrial form. In addition, the size of chloroplasts of the Kranz cells was 60–70% of that of the terrestrial form. These structural differences between the two forms may be related to the functional differences in their mechanisms of photosynthesis.Abbreviations KC
Kranz cell
- MC
mesophyll cell
- PSC
parenchyma sheath cell
- NAD-ME
NAD-malic enzyme
- VB
vascular bundle
This study was supported by Grants-in-Aid from the Ministry of Agriculture, Forestry and Fisheries of Japan (Integrated Research Program for the Use of Biotechnological Procedures for Plant Breeding) and from the Science and Technology Agency of Japan (Enhancement of Center-of-Excellence, the Special Coordination Funds for Promoting Science and Technology). 相似文献
12.
In its leaf blade, Arundinella hirta has unusual Kranz cells that lie distant from the veins (distinctive cells; DCs), in addition to the usual Kranz units composed of concentric layers of mesophyll cells (MCs) and bundle sheath cells (BSCs; usual Kranz cells) surrounding the veins. We examined whether chlorophyllous organs other than leaf blades—namely, the leaf sheath, stem, scale leaf, and constituents of the spike—also have this unique anatomy and the C4 pattern of expression of photosynthetic enzymes. All the organs developed DCs to varying degrees, as well as BSCs. The stem, rachilla, and pedicel had C4-type anatomy with frequent occurrence of DCs, as in the leaf blade. The leaf sheath, glume, and scale leaf had a modified C4 anatomy with MCs more than two cells distant from the Kranz cells; DCs were relatively rare. An immunocytochemical study of C3 and C4 enzymes revealed that all the organs exhibited essentially the same C4 pattern of expression as in the leaf blade. In the scale leaf, however, intense expression of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) occurred in the MCs as well as in the BSCs and DCs. In the leaf sheath, the distant MCs also expressed Rubisco. In Arundinella hirta, it seems that the ratio of MC to Kranz cell volumes, and the distance from the Kranz cells, but not from the veins, affects the cellular expression of photosynthetic enzymes. We suggest that the main role of DCs is to keep a constant quantitative balance between the MCs and Kranz cells, which is a prerequisite for effective C4 pathway operation. 相似文献
13.
Osuna L Coursol S Pierre JN Vidal J 《Biochemical and biophysical research communications》2004,315(2):428-433
We have shown previously that fibroblasts derived from fat or dermal tissue differ in their functional properties, such as proliferation rate and contractile properties. To study these differences further, two-dimensional electrophoresis (2D PAGE) was performed on proteins isolated from cultured subcutaneous fat and dermal fibroblasts. The 2D gels were screened for proteins that were differentially expressed in all donors (n = 5). Five protein spots were subjected to further analysis by mass spectrometry. Two proteins could be identified: brain acid soluble protein 1 (BASP1) and cellular retinoic acid binding protein-II (CRABP-II). CRABP-II is of interest in terms of re-epithelialisation and was clearly expressed in dermal fibroblasts but not in fat fibroblasts. Real time PCR was performed to confirm the 2D data on CRABP-II. The CRABP-II mRNA level was significantly increased in dermal tissue and cultured dermal fibroblasts compared to fat tissue and cultured fat-derived fibroblasts, respectively. The mode of action of CRABP-II in skin is to mediate retinoic acid activity. Retinoic acid is known to inhibit migration and to stimulate differentiation of keratinocytes. The expression of CRABP-II by dermal fibroblasts implicates a role for these fibroblasts in wound re-epithelialisation, in contrast to subcutaneous fat-derived fibroblasts. 相似文献
14.
L.-J. Ou W.-J. Li L.-F. Tian Z. Chen X.-J. Dai L.-X. Deng Y.-L. Qiu H. Li P. Liu M.-Z. Liang S. Luan L.-B. Chen 《Photosynthetica》2008,46(4):589-594
Biao 810S is a chlorina mutant of the thermosensitive genic male sterile (TGMS) rice. We compared photosynthetic characteristics of these two lines.
The contents of chlorophylls and carotenoids in Biao 810S were approximately half of those in 810S. However, the net photosynthetic
rate (P
N) of Biao 810S was higher than that of 810S under high irradiance or low concentration of carbon dioxide, and the photon quantum
efficiency was higher than that of 810S. The activity of ribulose-1,5-bisphosphate carboxylase/oxygenase in Biao 810S was
only 69.80 % of that in 810S, but the activities of phosphoenolpyruvate carboxylase and NADP-malic enzyme were 79.50 and 69.06 % higher than those of 810S, respectively, suggesting that
the efficiency of photon energy utilization in Biao 810S was enhanced by reduction of thermal dissipation and increase of
electron transfer rate to generate sufficient assimilation power for the dark reactions. Consequently, the increased activities
of C4 photosynthetic enzymes lead to more effective fixation of CO2 and the synergistic effect of light and dark reactions contributed to the higher P
N of Biao 810S. 相似文献
15.
16.
17.
18.
Species in the Laxa and Grandia groups of the genus Panicum are adapted to low, wet areas of tropical and subtropical America. Panicum milioides is a species with C3 photosynthesis and low apparent photorespiration and has been classified as a C3/C4 intermediate. Other species in the Laxa group are C3 with normal photorespiration. Panicum prionitis is a C4 species in the Grandia group. Since P. milioides has some leaf characteristics intermediate to C3 and C4 species, its photosynthetic response to irradiance and temperature was compared to the closely related C3 species, P. laxum and P. boliviense and to P. prionitis. The response of apparent photosynthesis to irradiance and temperature was similar to that of P. laxum and P. boliviense, with saturation at a photosynthetic photo flux density of about 1 mmol m-2 s-1 at 30°C and temperature optimum near 30°C. In contrast, P. prionitis showed no light saturation up to 2 mmol m-2 s-1 and an optimum temperature near 40°C. P. milioides exhibited low CO2 loss into CO2-free air in the light and this loss was nearly insensitive to temperature. Loss of CO2 in the light in the C3 species, P. laxum and P. boliviense, was several-fold higher than in P. milioides and increased 2- to 5-fold with increases in temperature from 10 to 40°C. The level of dark respiration and its response to temperature were similar in all four Panicum species examined. It is concluded that the low apparent photorespiration in P. milioides does not influence its response of apparent photosynthesis to irradiance and temperature in comparison to closely related C3 Panicum species.Abbreviations AP
apparent photosynthesis
- I
CO2 compensation point
- gl
leaf conductance; gm, mesophyll conductance
- PPFD
photosynthetic photon flux density
- PR
apparent photorespiration rate
- RuBPC
sibulose bisphosphate carboxylase 相似文献
19.
Russell K. Monson Robert O. Littlejohn Jr. George J. Williams III 《Photosynthesis research》1982,3(2):153-159
The quantum yield for CO2 uptake was measured in C3 and C4 monocot species from several different grassland habitats. When the quantum yield was measured in the presence of 21% O2 and 340 cm3 m-3 CO2, values were very similar in C3 monocots, C3 dicots, and C4 monocots (0.045–0.056 mole CO2 · mole-1 quanta absorbed). In the presence of 2% O2 and 800 cm3 m-3 CO2, enhancements of the quantum yield values occurred for the C3 plants (both monocots and dicots), but not for C4 monocots. A dependence of the quantum yield on leaf temperature was observed in the C3 grass, Agropyron smithii, but not in the C4 grass, Bouteloua gracilis, in 21% O2 and 340 cm3 m-3 CO2. At leaf temperatures between 22–25°C the quantum yield values were approximately equal in the two species. 相似文献
20.
The aquatic monocot Hydrilla verticillata (L.f.) Royle is a well-documented facultative C4 NADP-malic enzyme species in which the C4 and Calvin cycles operate in the same cell with the specific carboxylases confined to the cytosol and chloroplast, respectively. Several key components had already been characterized at the molecular level, thus the purpose of this study was to begin to identify other, less obvious, elements that may be necessary for a functional single-cell C4 system. Using differential display, mRNA populations from C3 and C4 H. verticillata leaves were screened and expression profiles compared. From this study, 65 clones were isolated and subjected to a customized macroarray analysis; 25 clones were found to be upregulated in C4 leaves. Northern and semi-quantitative RT-PCR analyses were used for confirmation. From these screenings, 13 C4 upregulated genes were identified. Among these one encoded a previously recognized C4 phosphoenolpyruvate carboxylase, and two encoded distinct pyruvate orthophosphate dikinase isoforms, new findings for H. verticillata. Genes that encode a transporter, an aminotransferase and two chaperonins were also upregulated. Twelve false positives, mostly housekeeping genes, were determined from the Northern/semi-quantitative RT-PCR analyses. Sequence data obtained in this study are listed in the dbEST database (DV216698 to DV216767). As a single-cell C4 system that lacks Kranz anatomy, a better understanding of how H. verticillata operates may facilitate the design of a transgenic C4 system in a C3 crop species.Srinath K. Rao and Hiroshi Fukayama contributed equally to this study. 相似文献