PEPC Activity Research Articles

Comparison of Water Stress and uv Radiation Effects on Induction of Cam and Antioxidative Defense in the Succulent Rosularia Elymaitica (Crassulaceae)

Growth and photosynthetic characteristics, inducibility of the CAM pathway and the functioning of the antioxidant defense system were investigated in Rosularia elymaitica (Crassulaceae) under drought and UV stresses. Drought did not substantially affect the growth of the plants, but it significantly reduced leaf thickness as well as osmotic potential, water potential and relative water content. In contrast, UV radiation treatment affected neither growth nor the water relations of leaves. Water limitation for 12 days caused a significant increase in nighttime PEPC and NAD-MDH activity and an increase in Δtitratable acidity relative to well-watered plants. The nighttime CO2 net assimilation rate increased significantly in drought-stressed plants but was still negative, resembling a C3-like pattern of gas exchange. Twenty days of UV treatment, increased Δtitratable acidity slightly and increased only daytime PEPC activity, and did not affect other parameters of carbon metabolism. As judged by maintenance of membrane integrity and stable amounts of H2O2 under UV stress, the antioxidant defense system effectively protected the plants against UV radiation. In contrast, oxidative stress occurred under severe drought stress (20 days of withholding water). Except for higher daytime APX activity in the UV-treated plants, enzyme activity in the control and in the drought- and UV-stressed plants did not show any diurnal fluctuation during 24 h. Temporal changes in Δtitratable acidity and ΔPEPC activity coincided closely with those of antioxidant enzymes; both started to increase after 12 days of drought stress. These results indicate that drought stress but not UV radiation induced the CAM-cycling pathway in R. elymaitica.

CRASSULACEAN ACID METABOLISM IN PHALAENOPSIS APHRODITE VAR. FORMOSA DURING DIFFERENT DEVELOPMENTAL STAGES

Mature Phalaenopsis is a typical CAM plant; whereas, no remarkable day/ night titratable acidity rhythm was detected at protocorm stage. The observation suggested that Phalaenopsis at its youth may undergo C3 photosynthesis pathway and lead to our hypothesis that there exists a photosynthetic transition from C3 to CAM during plant development. Phalaenopsis aphrodite var. formosa, the most important indigenous species in Taiwan, was used to analyze PEPC activity and gene expression related to such photosynthesis transition. There was no significant daily change in malate and PEPC activity from stage 0 to 2. Malate and PEPC activity showed a daily rhythm since stage 3. At stage 4, which the protocorm developed the first leaf, the typical daily CAM pattern of malate and PEPC activity was observed. Hence, stage 4 may be the photosynthesis transition period after leaf differentiation. However, the expression of PPC, encoded for PEPC, revealed a constitutional pattern in all developmental stages without day/night difference. These results suggest that PPC expression is not the key factor controlling the transformation of photosynthesis type, and genes responsible for activation of PEPC are our next targets for studying photosynthesis transition.

Characterization of bacterial-type phosphoenolpyruvate carboxylase expressed in male gametophyte of higher plants

BackgroundPhosphoenolpyruvate carboxylase (PEPC) is a critical enzyme catalyzing the β-carboxylation of phosphoenolpyruvate (PEP) to oxaloacetate, a tricarboxylic acid (TCA) cycle intermediate. PEPC typically exists as a Class-1 PEPC homotetramer composed of plant-type PEPC (PTPC) polypeptides, and two of the subunits were reported to be monoubiquitinated in germinating castor oil seeds. By the large-scale purification of ubiquitin (Ub)-related proteins from lily anther, two types of PEPCs, bacterial-type PEPC (BTPC) and plant-type PEPC (PTPC), were identified in our study as candidate Ub-related proteins. Until now, there has been no information about the properties of the PEPCs expressed in male reproductive tissues of higher plants.ResultsExpression analyses showed that lily BTPC (LlBTPC) and Arabidopsis BTPC (AtBTPC) were significantly expressed in pollen. The fusion protein AtBTPC-Venus localized in the cytoplasm of the vegetative cell (VC). Both LlBTPC and AtBTPC expression initiated after the last mitosis before pollen germination. Lily PTPC (LlPTPC) and monoubiquitinated LlPTPC (Ub-LlPTPC) remained at constant levels during pollen development. In late bicellular pollen of lily, LlBTPC forms a hetero-octameric Class-2 PEPC complex with LlPTPC to express PEPC activity.ConclusionOur results suggest that an LlBTPC:Ub-LlPTPC:LlPTPC complex is formed in the VC cytoplasm during late pollen development. Both LlBTPC and AtBTPC expression patterns are similar to the patterns of the appearance of storage organelles during pollen development in lily and Arabidopsis, respectively. Therefore, BTPC is thought to accelerate the metabolic flow for the synthesis of storage substances during pollen maturation. Our study provides the first characterization of BTPC in pollen, the male gametophyte of higher plants.

Synergic effect of salinity and CO2 enrichment on growth and photosynthetic responses of the invasive cordgrass Spartina densiflora.

Spartina densiflora is a C4 halophytic species that has proved to have a high invasive potential which derives from its clonal growth and its physiological plasticity to environmental factors, such as salinity. A greenhouse experiment was designed to investigate the synergic effect of 380 and 700 ppm CO2 at 0, 171, and 510 mM NaCl on the growth and the photosynthetic apparatus of S. densiflora by measuring chlorophyll fluorescence parameters, gas exchange and photosynthetic pigment concentrations. PEPC activity and total ash, sodium, potassium, calcium, magnesium, and zinc concentrations were determined, as well as the C/N ratio. Elevated CO2 stimulated growth of S. densiflora at 0 and 171 mM NaCl external salinity after 90 d of treatment. This growth enhancement was associated with a greater leaf area and improved leaf water relations rather than with variations in net photosynthetic rate (A). Despite the fact that stomatal conductance decreased in response to 700 ppm CO2 after 30 d of treatment, A was not affected. This response of A to elevated CO2 concentration might be explained by an enhanced PEPC carboxylation capacity. On the whole, plant nutrient concentrations declined under elevated CO2, which can be ascribed to the dilution effect caused by an increase in biomass and the higher water content found at 700 ppm CO2. Finally, CO2 and salinity had a marked overall effect on the photochemical (PSII) apparatus and the synthesis of photosynthetic pigments.

Changes of metabolic responses to direct and induced Fe deficiency of two Pisum sativum cultivars

The aim of this work was to investigate the effect of Fe deficiency (whether direct or bicarbonate-induced) on the glucose metabolism, by determining the activities of several enzymes (PK, PFK, G3PDH and G6PDH), together with other enzymes involved in the organic acids metabolism (PEPC, MDH, CS and ICDH) in two cultivars of Pisum sativum (Kelvedon and Lincoln, respectively tolerant and sensitive to Fe deficiency). Moreover, we studied the effects of Fe deficiency on iron reduction mechanism and proton extrusion (FCR and H +-ATPase activity, respectively), in addition to the concentrations in roots and exudates of some organic compounds (malate, citrate and phenols). Three treatments were used: (+Fe), complete medium (CM) containing 30 μM Fe; (−Fe), direct deficiency, CM without iron; (+Bic.), indirect deficiency, CM containing 30 μM Fe + lime. Our results showed that all glycolytic enzymes activities increased in the extracts of Fe-deficient roots of both cultivars when compared to the control. The above increases in the activity were particularly evident for Fe-deficient roots of Kelvedon. FCR and H +-ATPase activity was stimulated by Fe deficiency in both cultivars. PEPC activity increased by 254% in root extracts of Kelvedon particularly when grown in the absence of Fe. MDH, CS and ICDH activities showed a marked increase in the −Fe treated roots of both cultivars. The bicarbonate treatment resulted in a significant induction of root MDH and CS activities only in Kelvedon cultivar. In leaves, CS activity increased significantly in Fe deficient plants of both cultivars. However, Fe deficiency (whether direct or indirect) had no effect on the leaf activity of MDH and ICDH. Citrate concentration increased in both cultivars, particularly in Kelvedon (187% and 50% of the control, respectively in the −Fe and +bicarbonate treatments). Higher concentrations of phenols were observed in Fe-deficient roots of both cultivars than in the control plants. The increase in phenol concentration in roots was greater in Kelvedon than in Lincoln. We demonstrate that the metabolic responses of the two cultivars are differently affected by Fe deficiency both direct and induced by bicarbonate supply. The effective modulation of glycolytic and organic acid metabolisms may account for the better tolerance of Kelvedon when compared to Lincoln.

Phosphoenolpyruvate Carboxylase from C4 Leaves Is Selectively Targeted for Inhibition by Anionic Phospholipids

Phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) is an enzyme playing a crucial role in photosynthesis of C4 plants. Here, we identify anionic phospholipids as novel regulators that inhibit C4 PEPC activity and provide evidence that the enzyme partially localizes to membranes.

Metabolic‐dependent changes in plant cell redox power after ozone exposure

The tropospheric level of the phytotoxic air pollutant ozone has increased considerably during the last century, and is expected to continue to rise. Long-term exposure of higher plants to low ozone concentrations affects biochemical processes prior to any visible symptoms of injury. The current critical level of ozone used to determine the threshold for damaging plants (biomass loss) is still based on the seasonal sum of the external concentration above 40 nl.l(-1) (AOT40). Taking into account stomatal conductance and the internal capacity of leaf defences, a more relevant concept should be based upon the 'effective ozone flux', the balance between the stomatal flux and the intensity of cellular detoxification. The large decrease in the Rubisco/PEPc ratio reflects photosynthetic damage from ozone, and a large increase in activity of cytosolic PEPc, which allows increased malate production. Although the direct detoxification of ozone (and ROS produced from its decomposition) is carried out primarily by cell wall ascorbate, the existing level of this antioxidant is not sufficient to indicate the degree of cell sensitivity. In order to regenerate ascorbate, NAD(P)H is needed as the primary supplier of reducing power. It is hypothesised that increased activity of the catabolic pathways and associated shunts (glucose-6-phosphate dehydrogenase, NADP-dependent glyceraldehyde-3-phosphate dehydrogenase, isocitrate dehydrogenase and malic enzyme) can provide sufficient NAD(P)H to maintain intracellular detoxification. Thus, measurement of the level of redox power would contribute to determination of the 'effective ozone dose', serving ultimately to improve the ozone risk index for higher plants.

Reversible phosphorylation of photosynthetic PEP carboxylase: Studies on C4-leaf PP2A and recombinant PEPC-kinase from CAM-induced Mesembryanthemum crystallinum

The activity and allosteric properties of plant PEPC are controlled posttranslationally by specific reversible phosphorylation of a strictly conserved Ser residue near the N-terminus. This up/down-modulation is catalyzed by a transcriptionally regulated, seemingly dedicated Ser/Thr kinase (PpcK) and an opposing Ser/Thr phosphatase (PP2A). We have now partially purified and characterized the native form of this largely neglected PP2A from maize leaves using various chromatographic and affinity matrices, and C4 [32P]PEPC as substrate (Dong et al., 2001, Planta [in press]). The results indicate that the C4-leaf holoenzyme is analogous to yeast and mammalian PP2As in regards to its heterotrimeric structure (~170 kDa), comprised of a ~103-kDa core PP2Ac-A heterodimer complexed with a ~74-kDa B-type subunit, and its sensitivity to free Me2+ and various inhibitors, activators and anionic metabolites. Notably, this native PP2A (a) lacks any strict phosphoprotein specificity in that it dephosphorylates C4 PEPC, mammalian phosphorylase a, and casein in vitro, and (b) displays, at best, only modest light/dark effects in vivo on its apparent Mr, component core subunits, and activity against C4 PEPC-SerP. In addition, we will also report new findings on a recombinant form of CAM PpcK from M. crystallinum (Taybi et al., 2000, Plant Physiol.) produced as a highly soluble, active fusion with the ~55-kDa NusA carrier protein in E. coli. This NusA?PpcK fusion protein has been purified by sequential IMAC and FPLC, used for detailed analysis of its target-protein specificity and other kinetic properties, and cleaved on-bead by thrombin to yield free PpcK for antibody production.

Changes of photosynthetic activities of maize (Zea mays L.) seedlings in response to cadmium stress

Maize (Zea mays L.) seedlings were grown in nutrient solution culture containing 0, 5, and 20 μM cadmium (Cd) and the effects on various aspects of photosynthesis were investigated after 24, 48, 96 and 168 h of Cd treatments. Photosynthetic rate (P N) decreased after 48 h of 20 μM Cd and 96 h of 5μM Cd addition, respectively. Chl a and total Chl content in leaves declined under 48 h of Cd exposure. Chl b content decreased on extending the period of Cd exposure to 96 h. The maximum quantum efficiency and potential photosynthetic capacity of PSII, indicated by Fv/Fm and Fv/Fo, respectively, were depressed after 96 h onset of Cd exposure. After 48 h of 5μM Cd and 24 h of 20 μM Cd treatments, the activities of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.39) and phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) in the leaves started to decrease, respectively. We found that the limitation of photosynthetic capacity in Cd stressed maize leaves was associated with Cd toxicity on the light and the dark stages. However, Cd stress initially reduced the activities of Rubisco and PEPC and subsequently affected the PSII electron transfer, suggesting that the Calvin cycle reactions in maize plants are the primary target of the Cd toxic effect rather than PSII.

Activity of biochemical pH-stat enzymes in cereal root tips under oxygen deficiency

The activity of the enzymes of alcoholic and lactic-acid fermentation: pyruvate decarboxylase (PDC, EC 4.1.1.1), alcohol dehydrogenase (ADH, EC 1.1.1.1), lactate dehydrogenase (LDH, EC 1.1.1.27) and the enzymes of malic acid metabolism: phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.23), NAD-dependent malate dehydrogenase (NAD-MDH, EC 1.1.1.37), and NADP-dependent malic enzyme (NADP-ME, EC 1.1.1.40) involved in the operation of biochemical pH-stat was investigated in the root tips of wheat (Triticum aestivum L.) and rice (Oryza sativa L.) under hypoxia and anoxia. Exposures lasted for 6, 12, and 18 h. The most pronounced response was detected for the enzymes of alcoholic fermentation. The activation of ADH and PDC in wheat occurred only under hypoxia, whereas in rice it was detected both under hypoxia and anoxia. The activation of LDH in wheat occurred under hypoxia, and in rice, the activity of this enzyme was slightly enhanced. The activity of the enzymes of malic acid metabolism did not change except in wheat root tips under hypoxia when PEPC activity decreased and NADP-ME activity simultaneously rose. The role of biochemical pH-stat in the regulation of cytoplasmic pH in plant cells under oxygen deficit and the mechanisms for regulating the activities of enzymes involved in biochemical pH-stat are discussed as well as the interaction between biochemical pH-stat and other mechanisms maintaining pH of plant cells. The results are analyzed within a context of intracellular pH regulation.

Variability of metabolic responses and antioxidant defence in two lines of Medicago ciliaris to Fe deficiency

The aim of this work was to evaluate the effects of Fe deficiency on the activity of several metabolic enzymes (PEPC, PK, PFK, G6PDH and G3PDH), along with the function of the antioxidant enzymes (SK, SDH and PAL) in two lines of Medicago ciliaris, TN11.11 and TN8.7. Plants were grown in a greenhouse under controlled conditions. After germination and pre-treatment, plants were transferred for hydroponic culture. Three treatments were used: 30 μM Fe (+Fe), 0 μM Fe (−Fe) and 30 μM Fe + 10 mM NaHCO3 (+Bic.). Our results showed that all the enzymatic activities increased in extracts of Fe-deficient roots when compared to the control. The above increases in the activity were particularly evident for the bicarbonate-treated roots of TN11.11. PEPC activity was increased by 277% in TN11.11 plants with the addition of bicarbonate to the nutrient solution. Our results indicate also that, in the two lines of Medic, the activity of SK, SDH and PAL in leaves and roots were increased under Fe deficiency (either direct or induced by bicarbonate), to a greater extent in TN11.11 plants. Furthermore, a considerable increase in lipid peroxidation of roots and leaves of Fe-deficient plants was observed in TN8.7 when compared to TN11.11 plants. Our data suggest that the TN11.11 line is more effective in overcoming Fe deficiency than TN8.7. The tolerance of TN11.11 to Fe deficiency is related to its ability to modulate the carbohydrate metabolism and to increase secondary metabolism pathways.

Mechanisms of Aluminum Tolerance in Phosphoenolpyruvate Carboxylase Transgenic Rice

ABSTRACT Transgenic rice, in which C4-phosphoenolpyruvate carboxylase (PEPC) is overexpressed, was grown in hydroponic culture to evaluate its tolerance for high aluminum (Al) content in relation to organic acid synthesis and exudation into the rhizosphere. With regard to root growth in short-term experiments, PEPC-transgenic rice was more tolerant of Al than the wild type. At increased Al levels in the root medium, root tips of the transgenic rice accumulated less Al than those of the wild type. Aluminum-induced oxalate exudation from roots occurred at increased rates in the transgenic line. Overexpression of C4-PEPC drastically increased PEPC activity in leaves of the transgenic rice, causing higher organic acid concentrations in leaves and roots. The increased organic acid concentrations seemed to cause higher exudation rates of oxalate from roots, leading to a detoxification of Al in the root apoplast and rhizosphere.

Regulation of phosphoenolpyruvate carboxylase in PVYNTN-infected tobacco plants

The effect of viral infection on the regulation of phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) in Nicotiana tabacum L. leaves was studied. PEPC activity was 3 times higher in infected plant leaves compared to healthy plants. Activity of plant PEPC can be regulated, e.g., by de novo synthesis or reversible phosphorylation. The reason for the increase of PEPC activity as a consequence of PVY(NTN) infection was studied. The amount of PEPC determined by Western blot analysis or by relative estimation of PEPC mRNA by real-time PCR did not differ in control and PVY(NTN)-infected plants. Changes in posttranslational modification of PEPC by phosphorylation were evaluated by comparing activity of the native and the dephosphorylated enzyme. The infected plants were characterized by a higher decrease of the enzyme activity after its dephosphorylation, which indicated a higher phosphorylation level. Immunochemical detection of phosphoproteins by Western blot analysis showed a more intensive band corresponding to PEPC from the infected material. This strengthens the hypothesis of an infection-related phosphorylation, which could be part of the plant's response to pathogen attack. The physiological implications of the increase in PEPC activity during PVY(NTN) infection are discussed.

Impact of in vitro CO 2 enrichment and sugar deprivation on acclimatory responses of Phalaenopsis plantlets to ex vitro conditions

We assessed the effect of growth at either 400 μmol mol −1 (ambient) or 1000 μmol mol −1 (elevated) CO 2 and 0 g L −1 (deprivation) or 30 g L −1 (supplementation) sugar on morphological traits, photosynthetic attributes and intrinsic elements of the CAM pathway using the CAM orchid Phalaenopsis ‘Amaglade’. The growth of shoot (retarded) and root (induced) was differently affected by CO 2 enrichment and mixotrophic regime (+sugar). The Fv/Fm ratio was 14% more in CO 2-enriched treatment than at ambient level during in vitro growth. At elevated level of CO 2 and sugar treatment, the content of Chl( a + b), Chl a/ b and Chl/Car was enhanced while carotenoid content remained unaltered. During in vitro growth, gas-exchange analysis indicated that increased uptake of CO 2 accorded with the increased rate of transpiration and unchanged stomatal conductance at elevated level of CO 2 under both photo- and mixotrophic growth condition. At elevated level of CO 2 and sugar deprivation, activities of Rubisco (26.4%) and PEPC (74.5%) was up-regulated. Among metabolites, the content of sucrose and starch was always higher under CO 2 enrichment during both in vitro and ex vitro growth. Our results indicate that plantlets grown under CO 2 enrichment developed completely viable photosynthetic apparatus ready to be efficiently transferred to ex vitro condition that has far-reaching implications in micropropagation of Phalaenopsis.

Leaf succulence determines the interplay between carboxylase systems and light use during Crassulacean acid metabolism in Kalanchoë species*

The photosynthetic physiology of Crassulacean acid metabolism was investigated in two Kalanchoë species with differing leaf succulence. The magnitude of CAM was higher for the more succulent leaves of K. daigremontiana, compared to the less succulent leaves of K. pinnata. High succulence was related to low mesophyll conductance: K. pinnata was able to maximize diurnal carbon gain by the C(3) pathway, whereas increased succulence is associated with a higher commitment to the CAM cycle in K. daigremontiana. The Rubisco specificity factor, tau, determining selectivity for CO(2) over O(2), was similar for both species (approximately 88), and lower than that of Spinacea (approximately 95), but in contrast to C(4) plants, the Rubisco K(mCO(2)) (determined independently) was also lower in Kalanchoë spp. than in spinach. Differences in light use were related to the nature of the sink strength in each Phase of CAM, with PEPC activity resulting in low electron transport rates. Decarboxylation was marked by high, non-saturated rates of electron transport, with Rubisco activity and activation state increasing in both species during the course of the light period. The degree of succulence, and extent of CAM activity, was associated with a progressive inhibition of PSII photochemistry and potential Rubisco activity during the night in both species. Rubisco could be 'woken up' more rapidly in K. pinnata, whereas 45 min light acclimation was required for full recovery of electron transport and Rubisco activity in K. daigremontiana. Leaf morphology therefore seems to alter the expression of and dependence on CAM, but also the extent of co-regulation of carboxylase networks and light use capacity.

Treatment with salicylic acid decreases the effect of cadmium on photosynthesis in maize plants

The present study investigated the possible mediatory role of salicylic acid (SA) in protecting photosynthesis from cadmium (Cd) toxicity. Seeds of maize (Zea mays L., hybrid Norma) were sterilized and divided into two groups. Half of the seeds were presoaked in 500 microM SA solution for only 6h, after which both groups were allowed to germinate for 3d and were then grown for 14d in Hoagland solution at 22/18 degrees C in a 16/8-h light/dark period and 120 micromolm(-2)s(-1) PAR. All seedlings (without H(2)O and SA controls) were transferred to Cd-containing solutions (10, 15, and 25 microM) and grown for 14d. The rate of CO(2) fixation and the activity of ribulose 1,5-bisphosphate carboxylase (RuBPC, EC 4.1.1.39) and phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) were measured. Changes in the levels of several important parameters associated with oxidative stress, namely H(2)O(2) and proline production, lipid peroxidation, electrolyte leakage, and the activities of antioxidative enzymes (superoxide dismutase (SOD, EC 1.15.1.1), ascorbate peroxidase (APX, EC 1.11.1.11), catalase (CAT, EC 1.11.1.6), and guaiacol peroxidase (POD, EC 1.11.1.7)) were measured. Exposure of the plants to Cd caused a gradual decrease in the shoot and root dry weight accumulation, with the effect being most pronounced at 25 microM Cd. Seed pretreatment with SA alleviated the negative effect of Cd on plant growth parameters. The same tendency was observed for the chlorophyll level. The rate of CO(2) fixation was lower in Cd-treated plants, and the inhibition was partially overcome in SA-pretreated plants. A drop in the activities of RuBPC and PEPC was observed for Cd-treated plants. Pretreatment with SA alleviated the inhibitory effect of Cd on enzyme activity. Proline production and the rates of lipid peroxidation and electrolyte leakage increased in Cd-treated plants, whereas the values of these parameters were much lower in SA-pretreated plants. Treatment of plants with Cd decreased APX activity, but more than doubled SOD activity. Pretreatment with SA caused an increase in both APX and SOD activity, but caused a strong reduction in CAT activity. The data suggest that SA may protect cells against oxidative damage and photosynthesis against Cd toxicity.

The role of phosphoenolpyruvate carboxylase during C4 photosynthetic isotope exchange and stomatal conductance.

Phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) plays a key role during C(4) photosynthesis and is involved in anaplerotic metabolism, pH regulation, and stomatal opening. Heterozygous (Pp) and homozygous (pp) forms of a PEPC-deficient mutant of the C(4) dicot Amaranthus edulis were used to study the effect of reduced PEPC activity on CO(2) assimilation rates, stomatal conductance, and (13)CO(2) (Delta(13)C) and C(18)OO (Delta(18)O) isotope discrimination during leaf gas exchange. PEPC activity was reduced to 42% and 3% and the rates of CO(2) assimilation in air dropped to 78% and 10% of the wild-type values in the Pp and pp mutants, respectively. Stomatal conductance in air (531 mubar CO(2)) was similar in the wild-type and Pp mutant but the pp mutant had only 41% of the wild-type steady-state conductance under white light and the stomata opened more slowly in response to increased light or reduced CO(2) partial pressure, suggesting that the C(4) PEPC isoform plays an essential role in stomatal opening. There was little difference in Delta(13)C between the Pp mutant (3.0 per thousand +/- 0.4 per thousand) and wild type (3.3 per thousand +/- 0.4 per thousand), indicating that leakiness (), the ratio of CO(2) leak rate out of the bundle sheath to the rate of CO(2) supply by the C(4) cycle, a measure of the coordination of C(4) photosynthesis, was not affected by a 60% reduction in PEPC activity. In the pp mutant Delta(13)C was 16 per thousand +/- 3.2 per thousand, indicative of direct CO(2) fixation by Rubisco in the bundle sheath at ambient CO(2) partial pressure. Delta(18)O measurements indicated that the extent of isotopic equilibrium between leaf water and the CO(2) at the site of oxygen exchange () was low (0.6) in the wild-type and Pp mutant but increased to 0.9 in the pp mutant. We conclude that in vitro carbonic anhydrase activity overestimated as compared to values determined from Delta(18)O in wild-type plants.

Regulatory Properties of Phosphoenolpyruvate Carboxylase in Crassulacean Acid Metabolism Plants: Diurnal Changes in Phosphorylation State and Regulation of Gene Expression

Regulatory properties of Phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) in three CAM species, Kalanchoë pinnata, K. daigremontiana and Ananas comosus (pineapple) were examined. PEPC activity in the leaves of the three CAM species exhibited diurnal changes peaking during the first 2-h of darkness in Kalanchoë species and at midnight in pineapple, and then decreasing drastically until dawn. The oscillations of PEPC activity were far greater in Kalanchoë species than in pineapple. In the presence of 2 mM malate, the activity of PEPC decreased in all three CAM species, but the sensitivity of PEPC to malate was markedly different between pineapple and the Kalanchoë species. The malate sensitivity was 2- to 3-times higher in pineapple than in the Kalanchoë species during the dark period, but it was almost the same during the light period. PEPC in the three CAM species was phosphorylated only during the dark period. PEPC proteins were highly phosphorylated during the first 2-h of darkness in Kalanchoë species and at midnight in pineapple, and then they decreased drastically during the latter part of darkness. CAM-specific isoforms of PEPC in the leaves of the three CAM species contained a highly conserved phosphorylation site of Ser-11 at the N-terminus. These PEPC isoforms displayed diurnal changes in transcript abundance, with the peak of transcripts occurring during the dark period. The day/night changes in PEPC transcript abundance were mirrored by changes in the PEPC protein and corresponding enzyme activity over the diurnal cycle. These findings suggest that the diurnal regulation in PEPC activity is determined by the amount of PEPC protein as well as the posttranslational control in these CAM species.

Physiological impacts of modulating phosphoenolpyruvate carboxylase levels in leaves and seeds of Arabidopsis thaliana

Arabidopsis thaliana was transformed with a Sorghum C 4 PEPC cDNA under the control of the 35S promoter. Primary transformants were isolated and classified on a seed PEPC activity basis: A lines (two-fold increase) and B lines (four-fold increase). Primary transformants also expressed the C 4 PEPC in leaves (up to a 10-fold increase in activity). In seeds, ectopically expressed PEPC impacted on dry weight and total protein content that were increased by up to 30%. In the stable progeny of A and B lines, the increase in PEPC activity was amplified in seeds; however, a decrease in PEPC content and activity (inversely proportional to seed levels) was observed in maternal tissues (leaves and siliques). This was due to the absence of the exogenous C 4 PEPC and a partial decrease in the endogenous C 3 PEPC forms (mRNA, protein and activity). There was no apparent compensation by a PEPC hyperphosphorylation. In these plants, the anaplerotic pathway flux was altered (notably, a decrease in organic acids and glutamine levels) as judged by 1H NMR metabolite profiling. Whereas in the corresponding seeds (four- to eight-fold increase in PEPC activity) there was no increase in protein and seed mass, but a moderate decrease in lipid content was observed (compared to control plants). Under normal culture conditions, the transformants (primary and progeny) did not show any apparent growth phenotype or modification in seed production per plant. However, the A and B lines exhibited severe growth defects when salt stressed by LiCl.

Evolution of phosphoenolpyruvate carboxylase activity and lipid content during seed maturation of two spring rapeseed cultivars ( Brassica napus L.)

Phosphoenolpyruvate carboxylase (PEPc: EC 4.1.1.31) activity was monitored during seed maturation of two varieties (Hybridol and Pactol) of rapeseed ( Brassica napus L.), widely cultivated in Tunisia. In the Hybridol variety, PEPc activity did not exceed 5 μmol h −1 per gram of fresh weight (FW) during the first stages of maturation. It then highly increased to reach more than 30 μmol h −1 g −1/FW. On the contrary, in the Pactol variety, the evolution of PEPc activity showed a classical curve, i.e. an increase during the most active phase of lipid accumulation in maturating seeds, followed by a rapid decrease until the end of seed maturation. In both varieties, the seed oil was characterised by a high content of oleic acid (C 18:1), linoleic (C 18:2) and linolenic acids (C 18:3). Saturated fatty acids were also present, although decreasing with maturation course. The analysis of the triacylglycerols (TAG) showed that trioleoylglycerol (OOO) and dioleoyllinoleoylglycerol (OOL) were the major species (ca. 35% and ca. 25% of the total respectively). The evolution pattern of fatty acids and TAG contents was similar to that of PEPc activity. Taken together, our findings suggest that PEPc may be involved in fatty acid and triacylglycerol biosynthesis during seed maturation of both rapeseed varieties. To cite this article: K. Sebei et al., C. R. Biologies 329 (2006).