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1.
Maize is a typical C 4 plant of the NADP-malic enzyme type, and its high productivity is supported by the C 4 photosynthetic cycle, which concentrates atmospheric CO 2 in the leaves. The plant exhibits superior photosynthetic ability under high light and high temperature, but under cold conditions the photosynthetic rate is significantly reduced. Pyruvate orthophosphate dikinase (PPDK), a key enzyme of the C 4 pathway in maize, loses its activity below about 12 °C by dissociation of the tetramer and it is considered as one possible cause of the reduction in the photosynthetic rate of maize at low temperatures. To improve the cold stability of the enzyme, we introduced a cold-tolerant PPDK cDNA isolated from Flaveria
brownii into maize by Agrobacterium-mediated transformation. We obtained higher levels of expression by using a double intron cassette and a chimeric cDNA made from F. bidentis and F. brownii with a maximum content of 1mg/g fresh weight. In leaves of transgenic maize, PPDK molecules produced from the transgene were detected in cold-tolerant homotetramers or in heterotetramers of intermediate cold susceptibility formed with the internal PPDK. Simultaneous introduction of an antisense gene for maize PPDK generated plants in which the ratio of heterolologous and endogenous PPDK was greatly improved. Arrhenius plot analysis of the enzyme extracted from one such plant revealed that the break point was shifted about 3 °C lower than that of the wild type. 相似文献
2.
The nucleotide sequences of the complementary DNA of pyruvate, P i dikinase (PPDK) from Flaveria bidentis, a C 4 plant which possesses a cold-sensitive form of PPDK, and Flaveria brownii, a C 4-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. 相似文献
3.
Pyruvate orthophosphate dikinase (PPDK) was found in various immature seeds of C 3 plants (wheat, pea, green bean, plum, and castor bean), in some C 3 leaves (tobacco, spinach, sunflower, and wheat), and in C 4 (maize) kernels. The enzyme in the C 3 plants cross-reacts with rabbit antiserum against maize PPDK. Based on protein blot analysis, the apparent subunit size of PPDK from wheat seeds and leaves and from sunflower leaves is about 94 kdaltons, the same as that of the enzyme from maize, but is slightly less (about 90 kdaltons) for the enzyme from spinach and tobacco leaves. The amount of this enzyme per mg of soluble protein in C 3 seeds and leaves is much less than in C 4 leaves. PPDK is present in kernels of the C 4 plant, Zea mays in amounts comparable to those in C 4 leaves. Regulatory properties of the enzyme from C3 tissues (wheat) are similar to those of the enzyme from C4 leaves with respect to in vivo light activation and dark inactivation (in leaves) and in vivo cold lability (seeds and leaves). Following incorporation of 14CO2 by illuminated wheat pericarp and adjoining tissue for a few seconds, the labeled metabolites were predominantly products resulting from carboxylation of phosphoenolpyruvate, with lesser labeling of compounds formed by carboxylation of ribulose 1,5-bisphosphate and operation of the reductive pentose phosphate cycle of photosynthesis. PPDK may be involved in mechanisms of amino acid interconversions during seed development. 相似文献
4.
Pyruvate, orthophosphate dikinase (PPDK; E.C.2.7.9.1) is most well known as a photosynthetic enzyme in C 4 plants. The enzyme is also ubiquitous in C 3 plant tissues, although a precise non-photosynthetic C 3 function(s) is yet to be validated, owing largely to its low abundance in most C 3 organs. The single C 3 organ type where PPDK is in high abundance, and, therefore, where its function is most amenable to elucidation, are the developing seeds of graminaceous cereals. In this report, we suggest a non-photosynthetic function for C 3 PPDK by characterizing its abundance and posttranslational regulation in developing Oryza sativa (rice) seeds. Using primarily an immunoblot-based approach, we show that PPDK is a massively expressed protein during the early syncitial-endosperm/-cellularization stage of seed development. As seed development progresses from this early stage, the enzyme undergoes a rapid, posttranslational down-regulation in activity and amount via regulatory threonyl-phosphorylation (PPDK inactivation) and protein degradation. Immunoblot analysis of separated seed tissue fractions (pericarp, embryo + aleurone, seed embryo) revealed that regulatory phosphorylation of PPDK occurs in the non-green seed embryo and green outer pericarp layer, but not in the endosperm + aleurone layer. The modestly abundant pool of inactive PPDK (phosphorylated + dephosphorylated) that was found to persist in mature rice seeds was shown to remain largely unchanged (inactive) upon seed germination, suggesting that PPDK in rice seeds function in developmental rather than in post-developmental processes. These and related observations lead us to postulate a putative function for the enzyme that aligns its PEP to pyruvate-forming reaction with biosynthetic processes that are specific to early cereal seed development. 相似文献
6.
The protein content of seeds determines their nutritive value, downstream processing properties and market value. Up to 95% of seed protein is derived from amino acids that are exported to the seed after degradation of existing protein in leaves, but the pathways responsible for this nitrogen metabolism are poorly defined. The enzyme pyruvate,orthophosphate dikinase (PPDK) interconverts pyruvate and phospho enolpyruvate, and is found in both plastids and the cytosol in plants. PPDK plays a cardinal role in C 4 photosynthesis, but its role in the leaves of C 3 species has remained unclear. We demonstrate that both the cytosolic and chloroplastic isoforms of PPDK are up‐regulated in naturally senescing leaves. Cytosolic PPDK accumulates preferentially in the veins, while chloroplastic PPDK also accumulates in mesophyll cells. Analysis of microarrays and labelling patterns after feeding 13C‐labelled pyruvate indicated that PPDK functions in a pathway that generates the transport amino acid glutamine, which is then loaded into the phloem. In Arabidopsis thaliana, over‐expression of PPDK during senescence can significantly accelerate nitrogen remobilization from leaves, and thereby increase rosette growth rate and the weight and nitrogen content of seeds. This indicates an important role for cytosolic PPDK in the leaves of C 3 plants, and allows us to propose a metabolic pathway that is responsible for production of transport amino acids during natural leaf senescence. Given that increased seed size and nitrogen content are desirable agronomic traits, and that efficient remobilization of nitrogen within the plant reduces the demand for fertiliser applications, PPDK and the pathway in which it operates are targets for crop improvement. 相似文献
7.
The gene for C 4-pyruvate,orthophosphate dikinase (PPDK) from maize ( Zea mays) was cloned into an Escherichia coli expression vector and recombinant PPDK produced in E. coli cells. Recombinant enzyme was found to be expressed in high amounts (5.3 U purified enzyme-activity liter -1 of induced cells) as a predominantly soluble and active protein. Biochemical analysis of partially purified recombinant PPDK showed this enzyme to be equivalent to enzyme extracted from illuminated maize leaves with respect to (i) molecular mass, (ii) specific activity, (iii) substrate requirements, and (iv) phosphorylation/inactivation by its bifunctional regulatory protein.Abbreviations DTT-
dithiothreitol
- FPLC-
fast-protein liquid chromatography
- HAP-
hydroxyapatite
- IPTG-
isopropyl--thiogalactoside
- MOPS-
3-( N-morpholino)propanesulfonic acid
- PCR-
polymerase chain reaction
- PEP-
phospho enolpyruvate
- PMSF-
phenylmethylsufonyl fluoride
- PPDK-
pyruvate,orthophosphate dikinase
- RP-
regulatory protein 相似文献
8.
Pyruvate orthophosphate dikinase (PPDK) was detected in someC 3 plants, wheat, barley, rice and tobacco, by protein blottingusing an antibody against maize PPDK, although the amounts weremuch lesser than those of C 4 plants. The PPDK activity in immaturegrains of rice was specifically immunoprecipitated by the anti-(maize)PPDK antibody. The molecular weight of the subunit of PPDK inall tested C 3 plants was similar (ca. 95 kD) to that of maizePPDK, and the fragment patterns of the C 3 PPDKs in peptide mappingwere also similar to that of maize PPDK. These results suggestthat C 3 PPDKs have a primary structure similar to that of maizePPDK. In order to obtain information about the expression of PPDKin C 3 plants, changes in the enzyme activity and in the amountof PPDK protein were investigated during the greening of riceseedlings. PPDK, which was found in the etiolated seedlings,decreased temporarily in an early stage of greening and thenincreased. The mechanism of this variation is discussed.
1 To whom correspondence should be addressed. (Received December 9, 1986; Accepted March 12, 1987) 相似文献
11.
Polyadenylated RNA was isolated from maize leaves and translated in vitro. In agreement with a previous report by others, we found among the translation products a 110-kilodalton pyruvate orthophosphate dikinase (PPDK) precursor that is about 16 kilodaltons larger than the polypeptide isolated from cells. This maize PPDK precursor polypeptide was taken up from the translation product mixture by intact spinach chloroplasts and yielded a mature PPDK polypeptide (94 kilodaltons). The uptake and processing support the proposal that the extra 16-kilodalton size of the polypeptide from in vitro translation of maize leaf mRNA represents a transit sequence which is cleaved after its entry into chloroplasts. Moreover, these results provide additional evidence that in vivo in maize leaf cells PPDK polypeptide is synthesized in the cytoplasm and is transported into the chloroplasts. Location of PPDK in C3 plant leaves was investigated by immunochemical analysis. Intact chloroplasts were isolated from leaves of spinach, wheat, and maize. A protein blot of stromal protein in each case gave rise to bands corresponding to authentic PPDK polypeptide. This result indicates that PPDK is present in chloroplasts of C3 plant leaves as it is in the case of C4 plants. 相似文献
12.
In maize leaves, pyruvate, orthophosphate dikinase (PPDK) is deactivated in the dark and reactivated in the light. Studies in vitro using purified PPDK and a partially purified regulatory protein from maize confirmed previous reports correlating deactivation/reactivation with the reversible phosphorylation/dephosphorylation of a threonyl residue. By monitoring the stability of the exogenous 32P-labeled adenylate substrates during deactivation, we have firmly established ADP as the specific phosphate donor. In isolated maize leaf mesophyll protoplasts preilluminated with 32Pi, we observed a three- to fivefold higher PPDK activity in situ in the light, and a corresponding three- to fivefold higher level of phosphorylation of the 94-kDa PPDK protomer in the dark. HPLC-based phosphoamino acid analysis of PPDK purified from maize leaves of both light- and dark-adapted plants revealed the presence of P-serine. The inactive enzyme from dark-adapted plants (inactivated in vivo) also contained P-threonine. Total phosphate content of PPDK purified from leaves of light-adapted plants was approximately 0.5 mol/mol protomer, and 1.5 mol/mol protomer from leaves of dark-adapted plants. Since the difference between enzyme purified from light-adapted (active PPDK) and dark-adapted (inactive PPDK) plants is the presence of P-threonine in the latter, this suggests an inactivation stoichiometry in vivo of 1 mol P-threonine/mol 94-kDa protomer. These complementary studies with maize leaf PPDK in vitro, in situ, and in vivo provide convincing evidence for the dark/light regulation of this key C4-photosynthesis enzyme by reversible phosphorylation. 相似文献
14.
The high rates of photosynthesis and the carbon-concentrating mechanism (CCM) in C 4 plants are initiated by the enzyme phosphoenolpyruvate (PEP) carboxylase (PEPC). The flow of inorganic carbon into the CCM of C 4 plants is driven by PEPC’s affinity for bicarbonate ( KHCO3), which can be rate limiting when atmospheric CO 2 availability is restricted due to low stomatal conductance. We hypothesize that natural variation in KHCO3 across C 4 plants is driven by specific amino acid substitutions to impact rates of C 4 photosynthesis under environments such as drought that restrict stomatal conductance. To test this hypothesis, we measured KHCO3 from 20 C 4 grasses to compare kinetic properties with specific amino acid substitutions. There was nearly a twofold range in KHCO3 across these C 4 grasses (24.3 ± 1.5 to 46.3 ± 2.4 μm ), which significantly impacts modeled rates of C 4 photosynthesis. Additionally, molecular engineering of a low-HCO 3− affinity PEPC identified key domains that confer variation in KHCO3. This study advances our understanding of PEPC kinetics and builds the foundation for engineering increased-HCO 3− affinity and C 4 photosynthetic efficiency in important C 4 crops. 相似文献
15.
Three to four families of nuclear genes encode different isoforms of phospho enolpyruvate (PEP) carboxylase (PEPC): C 4-specific, C 3 or etiolated, CAM and root forms. C 4 leaf PEPC is encoded by a single gene ( ppc) in sorghum and maize, but multiple genes in the C 4-dicot Flaveria trinervia. Selective expression of ppc in only C 4-mesophyll cells is proposed to be due to nuclear factors, DNA methylation and a distinct gene promoter. Deduced amino acid sequences of C 4-PEPC pinpoint the phosphorylatable serine near the N-terminus, C 4-specific valine and serine residues near the C-terminus, conserved cysteine, lysine and histidine residues and PEP binding/catalytic sites. During the PEPC reaction, PEP and bicarbonate are first converted into carboxyphosphate and the enolate of pyruvate. Carboxyphosphate decomposes within the active site into Pi and CO 2, the latter combining with the enolate to form oxalacetate. Besides carboxylation, PEPC catalyzes a HCO 3
--dependent hydrolysis of PEP to yield pyruvate and Pi. Post-translational regulation of PEPC occurs by a phosphorylation/dephosphorylation cascade in vivo and by reversible enzyme oligomerization in vitro. The interrelation between phosphorylation and oligomerization of the enzyme is not clear. PEPC-protein kinase (PEPC-PK), the enzyme responsible for phosphorylation of PEPC, has been studied extensively while only limited information is available on the protein phosphatase 2A capable of dephosphorylating PEPC. The C 4
ppc was cloned and expressed in Escherichia coli as well as tobacco. The transformed E. coli produced a functional/phosphorylatable C 4 PEPC and the transgenic tobacco plants expressed both C 3 and C 4 isoforms. Site-directed mutagenesis of ppc indicates the importance of His 138, His 579 and Arg 587 in catalysis and/or substrate-binding by the E. coli enzyme, Ser 8 in the regulation of sorghum PEPC. Important areas for further research on C 4 PEPC are: mechanism of transduction of light signal during photoactivation of PEPC-PK and PEPC in leaves, extensive use of site-directed mutagenesis to precisely identify other key amino acid residues, changes in quarternary structure of PEPC in vivo, a high-resolution crystal structure, and hormonal regulation of PEPC expression.Abbreviations OAA
oxalacetate
- PEP
phospho enolpyruvate
- PEPC
PEP carboxylase
- PEPC-PK
PEPC-protein kinase
- PPDK
pyruvate, orthophosphate dikinase
- Rubisco
ribulose 1,5-bis-phosphate carboxylase/oxygenase
- CAM
Crassulacean acid metabolism 相似文献
16.
Soluble protein has been extracted from sections of wheat leaves, from base to tip, and the content of several key enzymes of photosynthetic carbon assimilation in each section has been determined by the protein blot method. In the first leaf, ribulose 1,5-bisphosphate carboxylase (RuBPC) (EC 4.1.1.39) in the basal 0 to 1 centimeter section is about 12% the level in the tip section, whereas phosphoenolpyruvate carboxylase (EC 4.1.1.31) is present in small amounts in the basal section and does not change much in the tip. Pyruvate orthophosphate dikinase (PPDK) (EC 2.7.9.1) first appears in the 4 to 6 centimeter section and increases gradually with development to 10-fold in the tip. Malic enzyme, NADP-dependent (EC 1.3.1.37) also appears in the 4 to 6 centimeter section but remains low to the tip. Fixation of 14CO2 by wheat leaf base sections resulted in 42% of total incorporation into malate and aspartate, indicating β-carboxylation, whereas in the tip section these labeled compounds were only 8% of the total. Although the amount of PPDK in wheat leaves is only 1 to 3% of that in maize leaves, this C3 PPDK may have a limited role in photosynthesis leading to formation of C4 compounds. The possibility of a further role, similar to that in C4 plants, but for intracellular carbon transport in wheat leaves is discussed. The presence of malic dehydrogenase, NADP-specific (EC 1.1.1.82) in wheat leaf chloroplasts was shown, a necessary though not sufficient condition for such a proposed role. Assuming each of the four enzymes associated with C4 carbon transport were fully active in vivo during photosynthesis, PPDK would still be rate limiting, even in the leaf tip where its activity is maximal. Possible evolutionary and breeding implications are discussed. 相似文献
17.
Eleocharis vivipara link, an amphibious leafless sedge, develops traits of C 4 photosynthesis and Kranz anatomy in the terrestrial form but develops C 3-like traits with non-Kranz anatomy when submerged. The cellular localization of C 3 and C 4 enzymes in the photosynthetic cells of the two forms was investigated by immunogold labeling and electron microscopy. The terrestrial form has mesophyll cells and three kinds of bundle sheath cell, namely, parenchyma sheath cells, non-chlorophyllous mestome sheath cells, and Kranz cells. Phosphoenol-pyruvate carboxylase (PEPCase) was present in the cytosol of both the mesophyll cells and the parenchyma sheath cells, with higher-density labeling in the latter, but not in the Kranz cells. Pyruvate, Pi dikinase (PPDK) was found at high levels in the chloroplasts of both the mesophyll cells and the parenchyma sheath cells with some-what stronger labeling in the latter. This enzyme was also absent from the Kranz cells. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) was found in the chloroplasts of all types of photosynthetic cell, but labeling was significantly less intense in the parenchyma sheath cells than in other types of cell. The submerged form also has three types of photosynthetic cell, as well as non-chlorophyllous mestome sheath cells, but it lacks the traits of Kranz anatomy as a consequence of modification of the cells. Rubisco was densely distributed in the chloroplasts of all the photosynthetic cells. However, PEPCase and PPDK were found in both the mesophyll cells and the parenchyma sheath cells but at lower levels than in the terrestrial form. These data reveal that the terrestrial form has a unique pattern of cellular localization of C 3 and C 4 enzymes, and they suggest that this pattern and the changes in the extent of accumulation of the various enzymes are the main factors responsible for the difference in photosynthetic traits between the two forms.Abbreviations CAM
crassulacean acid metabolism
- MC
meso phyll cell
- PSC
parenchyma sheath cell
- KC
Kranz cell
- PEP-Case
phosphoenolpyruvate carboxylase
- PPDK
pyruvate, Pi dikinase
- Rubisco
ribulose-1,5-bisphosphate carboxylase/oxygenase
- LS
large subunit
- RuBP
ribulose-1,5-bisphosphate
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). The author is grateful to Drs M. Matsuoka and S. Muto for providing the antisera and Dr. M. Samejima for his advice at the early stages of this study. 相似文献
18.
C 4 plants have two carboxylases which function in photosynthesis. One, phosphoenolpyruvate carboxylase (PEPC) is localized in mesophyll cells, and the other, ribulose bisphosphate carboxylase (RuBPC) is found in bundle sheath cells. In contrast, C 3 plants have only one photosynthetic carboxylase, RuBPC, which is localized in mesophyll cells. The expression of PEPC in C 3 mesophyll cells is quite low relative to PEPC expression in C 4 mesophyll cells. Two chimeric genes have been constructed consisting of the structural gene encoding β-glucuronidase (GUS) controlled by two promoters from C 4 (maize) photosynthetic genes: (i) the PEPC gene ( pepc) and (ii) the small subunit of RuBPC ( rbcS). These constructs were introduced into a C 3 cereal, rice. Both chimeric genes were expressed almost exclusively in mesophyll cells in the leaf blades and leaf sheaths at high levels, and no or very little activity was observed in other cells. The expression of both genes was also regulated by light. These observations indicate that the regulation systems which direct cell-specific and light-inducible expression of pepc and rbcS in C 4 plants are also present in C 3 plants. Nevertheless, expression of endogenous pepc in C 3 plants is very low in C 3 mesophyll cells, and the cell specificity of rbcS expression in C 3 plants differs from that in C 4 plants. Rice nuclear extracts were assayed for DNA-binding protein(s) which interact with a cis-regulatory element in the pepc promoter. Gel-retardation assays indicate that a nuclear protein with similar DNA-binding specificity to a maize nuclear protein is present in rice. The possibility that differences in pepc expression in a C 3 plant (rice) and C 4 plant (maize) may be the result of changes in cis-acting elements between pepc in rice and maize is discussed. It also appears that differences in the cellular localization of rbcS expression are probably due to changes in a trans-acting factor(s) required for rbcS expression. 相似文献
19.
First leaves and flag leaves of the wheat species Triticum aestivum cv Anza (6×), T. boeoticum Boiss (2×) L. were examined for content of pyruvate, orthophosphate dikinase (PPDK), phosphoenolpyruvate carboxylase (PEPC), and ribulose 1,5-bisphosphate carboxylase (RuBPC) by protein blot analyses using antibodies to maize leaf enzymes and by activity assays. In agreement with previous reports, the amount of RuBPC per mesophyll cell was about 3 times more in the hexaploid species, T. aestivum, than in the diploid species, T. boeoticum, both in first leaves and in flag leaves. In contrast, the level of PPDK polypeptide was nearly 3-fold higher per unit leaf area in the first leaf and 63% higher in the flag leaf of this diploid species compared to this hexaploid species. There was no significant difference in the levels of polypeptide and enzyme activity of PEPC between diploid and hexaploid wheat. Despite this significantly greater level of PPDK in the diploid species, the actual amount of PPDK could still supply only a limited amount of the enzyme activity necessary to provide phosphoenolpyruvate (PEP) for any putative intracellular C 4 carbon shuttle providing carbon to RuBPC. Thus, this difference in enzyme amount could not by itself account for the reported high rates of net photosynthesis at high light intensity in T. boeoticum. Together with reported anatomical differences between the diploid and hexaploid species, however, this biochemical difference may be of physiological importance. 相似文献
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