Changes in photosynthetic carbon flow in transgenic rice plants that express C4-type phosphoenolpyruvate carboxykinase from Urochloa panicoides.

A cDNA encoding phosphoenolpyruvate carboxykinase (PCK) of Urochloa panicoides (a PCK-type C4 plant) was expressed in rice (Oryza sativa cv Tsukinohikari) plants under the control of the promoter of a maize (Zea mays) gene for phosphoenolpyruvate carboxylase or pyruvate, orthophosphate dikinase with the transit peptide of the small subunit of Rubisco. Crude extracts prepared from the green leaves of transgenic plants had high PCK activity and the newly expressed PCK was localized in chloroplasts. In labeling experiments with (14)CO(2) up to 20% of the radioactivity was incorporated into 4C compounds (malate, oxaloacetate, and aspartate) in excised leaves of transgenic plants, as compared with about 1% in excised leaves of control plants. There was a positive correlation between PCK activity and the extent of labeling of 4C compounds. When L-[4-(14)C]malate was fed to excised leaves the extent of incorporation of radioactivity into sucrose was 3-fold greater in transgenic plants than in control plants and the level of radiolabeled aspartate was significantly lower in transgenic plants. These results indicate that the ectopic expression of PCK in rice chloroplasts was able partially to change the carbon flow in mesophyll cells into a C4-like photosynthetic pathway. Such a strategy appears to provide a possible method for enhancing the photosynthetic capacity of C3 plants.

Phosphoenolpyruvate carboxykinase in the C(4) monocot Urochloa panicoides is encoded by four differentially expressed genes.

Previous screening of a cDNA library of leaf poly(A(+)) RNA from Urochloa panicoides, a phosphoenolpyruvate carboxykinase (PCK)-type C(4) monocot, led to the characterization of cDNAs encoding the U. panicoides PCK subunit PCK1. A second PCK sequence, designated PCK2, has now been found by rescreening the library. The deduced PCK2 polypeptide is 626 residues in length, has a predicted molecular mass of 68,686 D, and is 96% identical to the deduced PCK1 sequence. Isolation and characterization of genomic DNA fragments revealed that the PCK1 and PCK2 genes are each closely linked to another PCK gene. These additional genes have been designated PCK3 and PCK4, respectively. In each case, the second gene is located upstream and in the same transcriptional orientation as the gene characterized through cDNA analysis. A reverse transcription-polymerase chain reaction assay was used to demonstrate that PCK1 and PCK2 transcripts predominate in leaves, whereas PCK3 and PCK4 transcripts predominate in roots. Moreover, accumulation of PCK1 and PCK2 transcripts is light dependent. Direct N-terminal sequencing of PCK polypeptides purified from leaves demonstrated that PCK2 is produced. These results strongly suggest that PCK1 and PCK2 are involved in the photosynthetic CO(2)-concentrating mechanism active in U. panicoides.

Identification of the amino acid residues responsible for cold tolerance in Flaveria brownii pyruvate,orthophosphate dikinase.

Pyruvate,orthophosphate dikinase (PPDK), an enzyme important in C4 photosynthesis, is typically a cold-sensitive enzyme. However, a cold-tolerant form of the enzyme has been isolated from the leaves of Flaveria brownii. Using an E. coli expression system and the PPDK cDNAs from F. brownii (cold-tolerant), F. bidentis (cold-sensitive) and maize (intermediately cold-tolerant), site-directed mutagenesis studies indicated that as few as three amino acids residues (of 880 residues) strongly influence the cold sensitivity of Flaveria PPDK. Gel filtration analysis of the PPDK expressed in E. coli showed that subunit association and cold tolerance are closely linked.

Identification of the amino acid residues responsible for cold tolerance in Flaveria brownii pyruvate,orthophosphate dikinase.

Pyruvate,orthophosphate dikinase (PPDK), an enzyme important in C4 photosynthesis, is typically a cold-sensitive enzyme. However, a cold-tolerant form of the enzyme has been isolated from the leaves of Flaveria brownii. Using an Escherichia coli expression system and the PPDK cDNAs from F. brownii (cold-tolerant), F. bidentis (cold-sensitive) and maize (intermediate cold tolerance), site-directed mutagenesis studies indicated that as few as three amino acids residues (of 880 residues) strongly influence the cold sensitivity of Flaveria PPDK. Gel filtration analysis of the PPDK expressed in E. coli showed that subunit association and cold tolerance are closely linked.

Molecular comparison of carbonic anhydrase from Flaveria species demonstrating different photosynthetic pathways.

During the evolution of C4 plants from C3 plants, both the function and intracellular location of carbonic anhydrase (CA) have changed. To determine whether these changes are due to changes at the molecular level, we have studied the cDNA sequences and the expression of CA from Flaveria species demonstrating different photosynthetic pathways. In leaf extracts from F. bidentis (C4), F. brownii (C4-like), F. linearis (C3-C4) and F. pringlei (C3), two polypeptides of M(r) 31 kDa and 35 kDa cross-reacted with anti-spinach CA antibodies. However, the relative labelling intensities of the two polypeptides differed depending on the species. Northern blot analysis indicated at least two CA transcripts are present in each Flaveria species with sizes ranging from 1.1 to 1.6 kb. Carbonic anhydrase cDNAs from all four Flaveria species studied encode an open reading frame for a polypeptide of 35-36 kDa. The amino acid sequences deduced from all four Flaveria cDNAs share at least 70% homology with the sequences of other dicot CAs. The F. bidentis (C4) CA sequence was found to be the least similar of the Flaveria proteins and, as most of the sequence dissimilarity was found in the first third of the CA molecule, these differences may be involved in the intracellular targeting of CA. A neighbour-joining tree inferred from CA amino acid sequences showed that the Flaveria CAs cluster with other dicot CAs forming a group distinct from those of monocot CAs and prokaryotic and Chlamydomonas periplasmic CAs.

Cold stability of pyruvate, orthophosphate dikinase of Flaveria brownii.

The nucleotide sequences of the complementary DNA of pyruvate, Pi dikinase (PPDK) from Flaveria bidentis, a C4 plant which possesses a cold-sensitive form of PPDK, and Flaveria brownii, a 'C4-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.

Isolation and sequence analysis of cDNAs encoding phosphoenolpyruvate carboxykinase from the PCK-type C4 grass Urochloa panicoides.

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 68,474 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.

Glutamine Induces the N-Dependent Accumulation of mRNAs Encoding Phosphoenolpyruvate Carboxylase and Carbonic Anhydrase in Detached Maize Leaf Tissue.

We have used detached leaves to study the N-dependent control of expression of phosphoenolpyruvate carboxylase (PEPC) and carbonic anhydrase (CA) genes in maize (Zea mays L. cv Golden Cross Bantam T51). Following supplementation with an N-source and zeatin, PEPC and CA mRNA levels increased in leaves detached from N-deficient maize plants. Addition of methionine sulfoximine (MSX), a specific inhibitor of glutamine synthetase, inhibited the nitrate-dependent increase of PEPC and CA mRNA but did not affect the glutamine-dependent increase of PEPC and CA mRNA levels. Glutamine levels in detached maize leaves treated with various N sources in the presence or absence of MSX correlated with the levels of PEPC and CA mRNA. We conclude that glutamine is the most likely effector for controlling the N-dependent expression of PEPC and CA in maize plants.

Cytokinin Is Required to Induce the Nitrogen-Dependent Accumulation of mRNAs for Phosphoenolpyruvate Carboxylase and Carbonic Anhydrase in Detached Maize Leaves.

Previous studies with intact maize (Zea mays L.) plants indicated that phosphoenolpyruvate carboxylase (PEPC) levels are controlled by nitrogen (N) availability and that this regulation is presumably at the transcriptional level (B. Sugiharto, K. Miyata, H. Nakamoto, H. Sasakawa, T. Sugiyama [1990] Plant Physiol 92: 963-969; B. Sugiharto, T. Sugiyama [1992] Plant Physiol 98: 1403-1408). In the present study, detached maize leaves were used to investigate further the mechanism of N-dependent regulation of gene expression in C(4) plants. PEPC and carbonic anhydrase (CA) mRNA levels decreased in leaves detached from maize plants. Addition of high nitrate did not prevent this decrease. However, the addition of zeatin to solutions bathing the cut ends of the detached leaves inhibited the decrease of PEPC and CA mRNA levels. Simultaneous addition of high nitrate and zeatin to leaves detached from N-deficient maize plants caused a large and rapid increase in PEPC and CA mRNA levels. Zeatin could be replaced by benzyladenine, but not by indoleacetic acid or abscisic acid. Both CA isozymes were effected and responded in an identical manner. We conclude that detached maize leaves provide an excellent experimental system to study the mechanism(s) of N-mediated regulation of PEPC and CA gene expression. However, zeatin is an essential component of this system.

Light Induction and the Effect of Nitrogen Status upon the Activity of Carbonic Anhydrase in Maize Leaves.

The regulation of carbonic anhydrase (CA) activity in maize (Zea mays L.) leaves by light and nitrogen nutrition was determined. CA activity increased by more than 100-fold in illuminated leaves and decreased in leaves placed in the dark; low levels of CA activity were observed in leaves illuminated with low light intensities. CA activity was reduced in plants grown under nitrogen deficiency and recovered only slowly when supplemented with nitrate. Parallel studies were conducted to follow the levels of phosphoenolpyruvate carboxylase. Experiments indicate that the level of CA and phosphoenolpyruvate carboxylase present in leaves may be controlled by similar mechanisms.

Carbonic anhydrase activity in leaves and its role in the first step of c(4) photosynthesis.

In C(4) plants carbonic anhydrase catalyzes the critical first step of C(4) photosynthesis, the hydration of CO(2) to bicarbonate. The maximum activity of this enzyme in C(4) leaf extracts, measured by H(+) production with saturating CO(2) and extrapolated to 25 degrees C, was found to be 3,000 to 10,000 times the maximum photosynthesis rate for these leaves. Similar activities were found in C(3) leaf extracts. However, the calculated effective activity of this enzyme at in vivo CO(2) concentrations was apparently just sufficient to prevent the rate of conversion of CO(2) to HCO(3) (-) from limiting C(4) photosynthesis. This conclusion was supported by the mass spectrometric determination of leaf carbonic anhydrase activities.

Spinach chloroplastic carbonic anhydrase: nucleotide sequence analysis of cDNA.

We have determined the nucleotide sequence of a cDNA encoding spinach (Spinacia oleracea) chloroplastic carbonic anhydrase (CA). The open reading frame encodes a protein consisting of a transit peptide and a mature CA protein with a predicted mass of 24, 116 daltons. This represents the first report of a nucleotide sequence of a plant CA.

Metabolite diffusion into bundle sheath cells from c(4) plants: relation to c(4) photosynthesis and plasmodesmatal function.

The present studies provide the first measurements of the resistance to diffusive flux of metabolites between mesophyll and bundle sheath cells of C(4) plants. Species examined were Panicum miliaceum, Urochloa panicoides, Atriplex spongiosa, and Zea mays. Diffusive flux of metabolites into isolated bundle sheath cells was monitored by following their metabolic transformation. Evidence was obtained that the observed rapid fluxes occurred via functional plasmodesmata. Diffusion constants were determined from the rate of transformation of limiting concentrations of metabolites via cytosolic enzymes with high potential velocities and favorable equilibrium constants. Values on a leaf chlorophyll basis ranged between 1 and 5 micromoles per minute per milligram of chlorophyll per millimolar gradient depending on the molecular weight of the metabolite and the source of bundle sheath cells. Diffusion of metabolites into these cells was unaffected by a wide variety of compounds including respiratory inhibitors, monovalent and divalent cations, and plant hormones, but it was interrupted by treatments inducing cell plasmolysis. The molecular weight exclusion limit for permeation of compounds into bundle sheath cells was in the range of 850 to 900. These cells provide an ideal system for the quantitative study of plasmodesmatal function.

Low bundle sheath carbonic anhydrase is apparently essential for effective c(4) pathway operation.

Bundle sheath cells from leaves of a variety of C(4) species contained little or no carbonic anhydrase activity. The proportion of total leaf carbonic anhydrase in extracts of bundle sheath cells closely reflected the apparent mesophyll cell contamination of bundle sheath cell extracts as measured by the proportion of the mesophyll cell marker enzymes phosphoenolpyruvate carboxylase and pyruvate,Pi dikinase. Values of about 1% or less of the total leaf activity were obtained for all three enzymes. The recorded bundle sheath carbonic anhydrase activity was compared with a calculated upper limit of carbonic anhydrase activity that would still permit efficient functioning of the C(4) pathway; that is, a carbonic anhydrase level allowing a sufficiently high steady state [CO(2)] to suppress photorespiration. Even before correcting for mesophyll cell contamination the activity in bundle sheath cell extracts was substantially less than the calculated upper limit of carbonic anhydrase activity consistent with effective C(4) function. The results accord with the notion that a deficiency of carbonic anhydrase in bundle sheath cells is vital for the efficient operation of the C(4) pathway.

Photosynthesis in phosphoenolpyruvate carboxykinase-type C4 plants: activity and role of mitochondria in bundle sheath cells.

Mitochondria from bundle sheath cells of the phosphoenolpyruvate carboxykinase-type C4 species Urochloa panicoides were shown to have metabolic properties consistent with a role in C4 photosynthesis predicted from earlier studies. The rate of O2 uptake in response to added malate plus ADP was at least five times the activity observed with NADH, glycine, or succinate. With malate plus ADP the O2 uptake rate averaged about 150 nmol O2 min-1 mg-1 protein, equivalent to about 0.6 mumol min-1 mg-1 of extracted chlorophyll. About half of this activity was apparently phosphorylation-linked with ADP/O2 ratios of about 4. Studies with electron transport inhibitors suggested that about 65% of this malate oxidation is cytochrome oxidase-terminated with a minor component mediated via the alternative oxidase. These mitochondria supported rapid rates of pyruvate production from malate and this activity was also stimulated by ADP but blocked by inhibitors of electron transport. Adding oxaloacetate increased pyruvate production but inhibited O2 uptake. The results were consistent with the notion that in this subgroup of C4 species mitochondrial-located NAD malic enzyme contributes substantially to total C4 acid decarboxylation. This enzyme is apparently also the primary source of NADH necessary to generate the ATP required for phosphoenolpyruvate carboxykinase-mediated oxaloacetate decarboxylation.

Photosynthesis in phosphoenolpyruvate carboxykinase-type C4 plants: pathways of C4 acid decarboxylation in bundle sheath cells of Urochloa panicoides.

The mechanism of C4 acid decarboxylation was studied in bundle sheath cell strands from Urochloa panicoides, a phosphoenolpyruvate carboxykinase (PCK)-type C4 plant. Added malate was decarboxylated to give pyruvate and this activity was often increased by adding ADP. Added oxaloacetate or aspartate plus 2-oxoglutarate (which produce oxaloacetate via aspartate aminotransferase) gave little metabolic decarboxylation alone but with added ATP there was a rapid production of PEP. For this activity ADP could replace ATP but only when added in combination with malate. In addition, the inclusion of aspartate plus 2-oxoglutarate with malate plus ADP often increased the rate of pyruvate production from malate by more than twofold. Experiments with respiratory chain inhibitors showed that the malate-dependent stimulation of oxaloacetate decarboxylation (PEP production) was probably due to ATP generated during the oxidation of malate in mitochondria. We could provide no evidence that photophosphorylation could serve as an alternative source of ATP for the PEP carboxykinase reaction. We concluded that both PEP carboxykinase and mitochondrial NAD-malic enzyme contribute to C4 acid decarboxylation in these cells, with the required ATP being derived from oxidation-linked phosphorylation in mitochondria.

Photosynthesis in phosphoenolpyruvate carboxykinase-type C4 plants: photosynthetic activities of isolated bundle sheath cells from Urochloa panicoides.

A method has been developed for rapidly preparing bundle sheath cell strands from Urochloa panicoides, a phosphoenolpyruvate (PEP) carboxykinase-type C4 plant. These cells catalyzed both HCO3(-)- and oxaloacetate-dependent oxygen evolution; oxaloacetate-dependent oxygen evolution was stimulated by ATP. For this activity oxaloacetate could be replaced by aspartate plus 2-oxoglutarate. Both oxaloacetate- and aspartate plus 2-oxoglutarate-dependent oxygen evolution were accompanied by PEP production and both were inhibited by 3-mercaptopicolinic acid, an inhibitor of PEP carboxykinase. The ATP requirement for oxaloacetate- and aspartate plus 2-oxoglutarate-dependent oxygen evolution could be replaced by ADP plus malate. The increased oxygen evolution observed when malate plus ADP was added with oxaloacetate was accompanied by pyruvate production. These results are consistent with oxaloacetate being decarboxylated via PEP carboxykinase. We suggest that the ATP required for oxaloacetate decarboxylation via PEP carboxykinase may be derived by phosphorylation coupled to malate oxidation in mitochondria. These bundle sheath cells apparently contain diffusion paths for the rapid transfer of compounds as large as adenine nucleotides.

Form of inorganic carbon involved as a product and as an inhibitor of c(4) Acid decarboxylases operating in c(4) photosynthesis.

These studies demonstrated that CO(2) rather than HCO(3) (-) is the inorganic carbon metabolite produced by the C(4) acid decarboxylases involved in C(4) photosynthesis (chloroplast located NADP malic enzyme, mitochondrial NAD malic enzyme, and cytosolic phosphoenolpyruvate [PEP] carboxykinase). The effect of varying CO(2) or HCO(3) (-) as a substrate for the carboxylation reaction catalyzed by these enzymes or as inhibitors of the decarboxylation reaction was also determined. The K(m)CO(2) was 1.1 millimolar for NADP malic enzyme and 2.5 millimolar for PEP carboxykinase. For these two enzymes the velocity in the carboxylating direction was substantially less than for the decarboxylating direction even with CO(2) concentrations at the upper end of the range of expected cellular levels. Activity of NAD malic enzyme in the carboxylating direction was undetectable. The decarboxylation reaction of all three enzymes was inhibited by added HCO(3) (-). For NADP malic enzyme CO(2) was shown to be the inhibitory species but PEP carboxykinase and NAD malic enzyme were apparently inhibited about equally by CO(2) and HCO(3) (-).