PEPC Activity Research Articles

Ozone exposure and flux-based response functions for photosynthetic traits in wheat, maize and poplar

Ozone exposure- and dose-response relationships based on photosynthetic leaf traits (CO2 assimilation, chlorophyll content, Rubisco and PEPc activities) were established for wheat, maize and poplar plants grown in identical controlled conditions, providing a comparison between crop and tree species, as well as between C3 and C4 plants. Intra-specific variability was addressed by comparing two wheat cultivars with contrasting ozone tolerance. Depending on plant models and ozone levels, first-order, second-order and segmented linear regression models were used to derive ozone response functions. Overall, flux-based functions appeared superior to exposure-based functions in describing the data, but the improvement remained modest. The best fit was obtained using the POD0.5 for maize and POD3 for poplar. The POD6 appeared relevant for wheat, although intervarietal differences were found. Our results suggest that taking into account the dynamics of leaf antioxidant capacity could improve current methods for ozone risk assessment for plants.

Phosphoenolpyruvate Carboxylase in Arabidopsis Leaves Plays a Crucial Role in Carbon and Nitrogen Metabolism.

Phosphoenolpyruvate carboxylase (PEPC) is a crucial enzyme that catalyzes an irreversible primary metabolic reaction in plants.Previous studies have used transgenic plants expressing ectopic PEPC forms with diminished feedback inhibition to examine the role of PEPC in carbon and nitrogen metabolism. To date, the in vivo role of PEPC in carbon and nitrogen metabolism has not been analyzed in plants. In this study, we examined the role of PEPC in plants, demonstrating that PPC1 and PPC2 were highly expressed genes encoding PEPC in Arabidopsis (Arabidopsis thaliana) leaves and that PPC1 and PPC2 accounted for approximately 93% of total PEPC activity in the leaves. A double mutant, ppc1/ppc2, was constructed that exhibited a severe growth-arrest phenotype. The ppc1/ppc2 mutant accumulated more starch and sucrose than wild-type plants when seedlings were grown under normal conditions. Physiological and metabolic analysis revealed that decreased PEPC activity in the ppc1/ppc2 mutant greatly reduced the synthesis of malate and citrate and severely suppressed ammonium assimilation. Furthermore, nitrate levels in the ppc1/ppc2 mutant were significantly lower than those in wild-type plants due to the suppression of ammonium assimilation. Interestingly, starch and sucrose accumulation could be prevented and nitrate levels could be maintained by supplying the ppc1/ppc2 mutant with exogenous malate and glutamate, suggesting that low nitrogen status resulted in the alteration of carbon metabolism and prompted the accumulation of starch and sucrose in the ppc1/ppc2 mutant. Our results demonstrate that PEPC in leaves plays a crucial role in modulating the balance of carbon and nitrogen metabolism in Arabidopsis.

Transformation of Barnyardgrass ( Echinochloa crusgalli ) Root Type Phosphoenolpyruvate Carboxylase Gene into Rice ( Oryza sativa ) Plants and Their Effects on Photosynthesitic Gas Exchange

Barnyardgrass(Echinochloa crusgalli) is a C4 weed commonly found in rice field. To fully utilize the photosynthestic potential of Barnyardgrass C4 gene, we transformed Barnyardgrass root Phosphoenolpyruvate Carboxylase gene into rice plant with vectors contained promoters of Ubiqitin gene and Rubisco small unit gene by Agrobactirium- mediated transformation. Both marker genes Hygr and ppc were detected by PCR in regenerated plants. RT-PCR and Western blot analysis confirmed that the ppc gene was incorporated into rice plant and expressed with stable transcripts and proteins. PEPC activity as measured in most of the transgenic rice plants was higher than that in control, being up to 5.85-fold of that in untransformed rice. At T0 generation, net photosynthetic rate(Pn) in most of transgenic rice plants was 20.00% higher than that in untransformed rice, with the highest increase of 47.16%. Water utilization efficiency(WUE) in transgenic rice was also improved. At T6 generation, PEPC activity and Pn of transgenic lines remained higher than those of the wild type. These indicate that over-expressing C3 Eppc gene also can improve rice photosynthesis.

Expression and knockdown of the PEPC1 gene affect carbon flux in the biosynthesis of triacylglycerols by the green alga Chlamydomonas reinhardtii.

The regulation of lipid biosynthesis is important in photosynthetic eukaryotic cells. This regulation is facilitated by the direct synthesis of fatty acids and triacylglycerol (TAG), and by other controls of the main carbon metabolic pathway. In this study, knockdown of the mRNA expression of the Chlamydomonas phosphoenolpyruvate carboxylase isoform 1 (CrPEPC1) gene by RNA interference increased TAG level by 20% but decreased PEPC activities in the corresponding transgenic algae by 39-50%. The decrease in CrPEPC1 expression increased the expression of TAG biosynthesis-related genes, such as acyl-CoA:diacylglycerol acyltransferase and phosphatidate phosphatase. Conversely, CrPEPC1 over-expression decreased TAG level by 37% and increased PEPC activities by 157-184%. These observations suggest that the lipid content of algal cells can be controlled by regulating the CrPEPC1 gene.

Preliminary Study on the Physiological Characteristics of Transgenic Wheat with Maize C4-pepcGene in Field Conditions

To explore the physiological characteristics of the pepc gene in transgenic wheat (Triticum aestivum) plants, PEPC activities in various organs of T3 plants were analyzed at Feekes 6.0, Feekes 10.3 and Feekes 11.1, and compared to control, untransformed wheat cultivar Zhoumai 19. Net photosynthetic rates (Pn) in leaves were also measured at the same stages. At Feekes 11.1, both transgenic and control plants were treated with DCDP. Yield traits were surveyed after harvest. The results indicated that Pn and PEPC activity in the flag leaf of transgenic wheat were significantly higher than those of the control at different stages. At Feekes 10.3, Pn reached the highest value at 28.2 μmol m−2 s−1 and PEPC activity reached the highest value at 104.6 μmol h−1 mg−1. Both factors significantly increased by 21% compared to the control at Feekes 11.1. PEPC activity in the flag leaf of transgenic plants was significantly higher than that of non-leaf organs. Pn of transgenic plants was greatly reduced after DCDP treatment. In the flag leaf of transgenic wheat, Pn was significantly correlated to PEPC activities at 0.01 probability level with a correlation coefficient of 0.8957**. The yield traits of transgenic line 1-27-3, such as 1000-grain weight, single spike weight and harvest index were higher than those of the control. Additionally, the spike weight of 1-27-3 showed an increase of approximately 9.5% compared to the control. These results indicated that the expression of maize (Zea mays) pepc gene was different across various organs of transgenic wheat and across every growth stage. Therefore, we conclude that introducing maize pepc gene into wheat plants can increase their Pn and improve production.

Leaf anatomical traits which accommodate the facultative engagement of crassulacean acid metabolism in tropical trees of the genus Clusia

Succulence and leaf thickness are important anatomical traits in CAM plants, resulting from the presence of large vacuoles to store organic acids accumulated overnight. A higher degree of succulence can result in a reduction in intercellular air space which constrains internal conductance to CO2. Thus, succulence presents a trade-off between the optimal anatomy for CAM and the internal structure ideal for direct C3 photosynthesis. This study examined how plasticity for the reversible engagement of CAM in the genus Clusia could be accommodated by leaf anatomical traits that could facilitate high nocturnal PEPC activity without compromising the direct day-time uptake of CO2 via Rubisco. Nine species of Clusia ranging from constitutive C3 through C3/CAM intermediates to constitutive CAM were compared in terms of leaf gas exchange, succulence, specific leaf area, and a range of leaf anatomical traits (% intercellular air space (IAS), length of mesophyll surface exposed to IAS per unit area, cell size, stomatal density/size). Relative abundances of PEPC and Rubisco proteins in different leaf tissues of a C3 and a CAM-performing species of Clusia were determined using immunogold labelling. The results indicate that the relatively well-aerated spongy mesophyll of Clusia helps to optimize direct C3-mediated CO2 fixation, whilst enlarged palisade cells accommodate the potential for C4 carboxylation and nocturnal storage of organic acids. The findings provide insight on the optimal leaf anatomy that could accommodate the bioengineering of inducible CAM into C3 crops as a means of improving water use efficiency without incurring detrimental consequences for direct C3-mediated photosynthesis.

Temperature Modulation of the Activity and Malate Inhibition of the Phosphoenolpyruvate Carboxylase from Leaves of Alternanthera pungens, Compared to that of Lycopersicom esculentum

Temperature caused marked modulation of phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) in leaf discs of Alternanthera pungens (C4 plant) as well as Lycopersicon esculentum (C3 species). The optimal incubation temperature for PEPC activity in A. pungens was 45 °C compared to 30 °C in L. esculentum. A. pungens lost nearly 61% of PEPC activity on exposure to a low temperature of 15 °C, compared to only about a 33% loss in the case of L. esculentum. The C4 enzyme was less sensitive to supra-optimal temperature and more sensitive to sub-optimal temperature than that of the C3 species. Further as the temperature was raised from 15 °C to 50 °C, there was a sharp decrease in malate sensitivity of PEPC. The extent of such a decrease in C4 plants was more than that in C3 species. Arrhenius plots that were constructed by plotting the activity of PEPC against the reciprocal of temperature in the absence or presence of malate exhibited abrupt changes or “break-points” at only one point of 17oC in A. pungens while at two points corresponding 17oC and 27oC in case of L. esculentum. The activation energy of PEPC from A. pungens was less compared to that of L. esculentum in the temperature range of 10 to 27oC. However, the activation energy of PEPC from A. pungens was less than that of L. esculentum above the temperature of 27oC. The activation energy increased by 2 to 4 fold at temperatures below 17oC, in case of both A. pungens and L. esculentum. Thus, our results show the activity and malate sensitivity of PEPC can be influenced in relation to high temperature tolerance of C4 plants, which can be physiologically significant.

Recombinant expression and functional analysis of a Chlamydomonas reinhardtii bacterial-type phosphoenolpyruvate carboxylase gene fragment

To investigate the function of a bacterial-type phosphoenolpyruvate carboxylase (PEPC2) derived from photosynthetically-grown Chlamydomonas reinhardtii, a fragment of the pepc2 gene was cloned and expressed in Escherichia coli. After optimal induction for 6 h, PEPC activity in the reverse mutant was lower than wild type (0.9 vs. 1.7 U/mg protein), and soluble protein was also lower than wild type (119 vs. 186 mg/g dry wt). In contrast, the total lipid content was increased from 56 (in wild type) to 71 mg/g dry wt, despite the growth rate being slightly diminished. The changes in PEPC activity, soluble protein and total lipid in the forward mutant were the opposite (2.4 U/mg, 230 mg/g, and 44 mg/g dry wt, respectively). Together, these data indicate that PEPC may function as a metabolic pivot in the regulation of protein and lipid accumulation in this alga.

Phosphoenolpyruvate carboxylase, NADP-malic enzyme, and pyruvate, phosphate dikinase are involved in the acclimation of Nicotiana tabacum L. to drought stress

Drought stress is one of the most frequent forms of abiotic stresses, which occurs under condition of limited water availability. In this work, the possible participation of phosphoenolpyruvate carboxylase (EC 4.1.1.31; PEPC), NADP-malic enzyme (EC 1.1.1.40; NADP-ME), and pyruvate, phosphate dikinase (EC 2.7.9.1; PPDK) in response to drought of tobacco plants (Nicotiana tabacum L., cv. W38) was investigated. Enzyme specific activities in tobacco leaves of drought stressed plants were significantly increased after 11 days of stress, PEPC 2.3-fold, NADP-ME 3.9-fold, and PPDK 2.7-fold compared to control plants. The regulation of PEPC and NADP-ME activities were studied on transcriptional level by the quantitative RT PCR and on translational level – immunochemically. The amount of NADP-ME protein and transcription of mRNA for chloroplastic NADP-ME isoform were increased indicating their enhanced synthesis de novo. On the other hand, mRNA for cytosolic isoform of NADP-ME was decreased. The changes in PEPC protein and PEPC mRNA were not substantial. Therefore regulation of PEPC activity by phosphorylation was evaluated and found to be involved in the stress response. During recovery, activities of the tested enzymes returned close to their basal levels.

Biochemical and biophysical CO2 concentrating mechanisms in two species of freshwater macrophyte within the genus Ottelia (Hydrocharitaceae)

Two freshwater macrophytes, Ottelia alismoides and O. acuminata, were grown at low (mean 5μmolL(-1)) and high (mean 400μmolL(-1)) CO2 concentrations under natural conditions. The ratio of PEPC to RuBisCO activity was 1.8 in O. acuminata in both treatments. In O. alismoides, this ratio was 2.8 and 5.9 when grown at high and low CO2, respectively, as a result of a twofold increase in PEPC activity. The activity of PPDK was similar to, and changed with, PEPC (1.9-fold change). The activity of the decarboxylating NADP-malic enzyme (ME) was very low in both species, while NAD-ME activity was high and increased with PEPC activity in O. alismoides. These results suggest that O. alismoides might perform a type of C4 metabolism with NAD-ME decarboxylation, despite lacking Kranz anatomy. The C4-activity was still present at high CO2 suggesting that it could be constitutive. O. alismoides at low CO2 showed diel acidity variation of up to 34μequiv g(-1) FW indicating that it may also operate a form of crassulacean acid metabolism (CAM). pH-drift experiments showed that both species were able to use bicarbonate. In O. acuminata, the kinetics of carbon uptake were altered by CO2 growth conditions, unlike in O. alismoides. Thus, the two species appear to regulate their carbon concentrating mechanisms differently in response to changing CO2. O. alismoides is potentially using three different concentrating mechanisms. The Hydrocharitaceae have many species with evidence for C4, CAM or some other metabolism involving organic acids, and are worthy of further study.

Physiological Characteristics of Delaying Leaf Senescence in Maize Inbred Lines Tolerant to Potassium Deficiency

A field experiment was conducted using two maize inbred lines with significant difference in potassium sensitivity to investigate the effects of low potassium stress at grain filling stage on leaf stay-green,chlorophyll contents,photosynthetic gas exchange and chlorophyll fluorescence parameters,and various antioxidant enzymes activities related with senescence.The results showed that,under low potassium stress,the characteristics of leaf stay-green of 90-21-3 were better than that of D937.In addition to changes in stay-green,it was found that decreased more strongly in D937,the photosynthetic pigment level in 90-21-3 was remained nearly normal under K deficiency conditions.Moreover,compared with D937,90-21-3 showed a lower stomatal restriction and a higher electronic transition capacity after accelerated ageing by K deficiency.FPSII,Fv,Fm in 90-21-3 were higher than in D937,while Fo was lower than those in D937.In addition,under low potassium stress,activities of PEPC and NADK were less decreased and photo system was less damaged in 90-21-3.And the content of malondialdehyde(MDA)was lower in 90-21-3 than in D937 under low potassium stress.The activities of superoxide dismutase(SOD),peroxidase(POD),and catalase(CAT)in ear leaf of 90-21-3 were obviously higher than those of D937 under low potassium stress,indicating that the antioxidant enzymes could eliminate reactive oxygen species,inhibit the membrane lipid peroxidation and decrease the content of MDA more efficiently in 90-21-3.Therefore,compared with D937,the leaf senescence of 90-21-3 was relatively slower,which could prolong the functional period of leaves.The activity of NADK could provide NADP as electronic receptors for photoreaction,at the same time the increased activity of PEPC led to more effective fixation of CO2,and the synergistic effect of light and dark reactions contributed to the higher photosynthetic rate and yield in 90-21-3.

Spatial division of phosphoenolpyruvate carboxylase and nitrate reductase activity and its regulation by cytokinins in CAM-induced leaves of Guzmania monostachia (Bromeliaceae)

Crassulacean acid metabolism (CAM) is a physiological adaptation of plants that live in stress environment conditions. A good model of CAM modulation is the epiphytic bromeliad, Guzmania monostachia, which switches between two photosynthetic pathways (C3–CAM) in response to different environmental conditions, such as light stress and water availability. Along the leaf length a gradient of acidity can be observed when G. monostachia plants are kept under water deficiency. Previous studies showed that the apical portions of the leaves present higher expression of CAM, while the basal regions exhibit lower expression of this photosynthetic pathway. The present study has demonstrated that it is possible to induce the CAM pathway in detached leaves of G. monostachia kept under water deficit for 7 d. Also, it was evaluated whether CAM expression can be modulated in detached leaves of Guzmania and whether some spatial separation between NO3− reduction and CO2 fixation occurs in basal and apical portions of the leaf. In addition, we analyzed the involvement of endogenous cytokinins (free and ribosylated forms) as possible signal modulating both NO3− reduction and CO2 fixation along the leaf blade of this bromeliad. Besides demonstrating a clear spatial and functional separation of carbon and nitrogen metabolism along G. monostachia leaves, the results obtained also indicated a probable negative correlation between endogenous free cytokinins – zeatin (Z) and isopentenyladenine (iP) – concentration and PEPC activity in the apical portions of G. monostachia leaves kept under water deficit. On the other hand, a possible positive correlation between endogenous Z and iP levels and NR activity in basal portions of drought-exposed and control leaves was verified. Together with the observations presented above, results obtained with exogenous cytokinins treatments, strongly suggest that free cytokinins might act as a stimulatory signal involved in NR activity regulation and as a negative regulator of PEPC activity in CAM-induced leaves of G. monostachia during a diel cycle.

Bacterial-type PEPC genes identification and expression in developing seeds from four oil crops

Bacterial-type PEPC genes were cloned from four oil crops(rape,soybean,peanut and sesame).Their sequences of conserved domains(BOX I to BOX IV) and PEPC activity sites were characterized.The genes included Bna10093361,Bna1009749 and Bna10093360 from Brassica napus,Glyma10g34970.1,Glyma01g22840.1 and Glyma02g14500.1 from Glycine max,SIN1018296 from Sesamum indicum,AhPPC5 from Arachis hypogaea.The 8 genes were commonly composed of 19 to 21 introns,and contained one unique and highly divergent insertion of about 356 to 600bp.PEPC sequences had R/KNTG in C-terminal tetrapeptide,but lacked the phosphorylation motif at N terminus.Results from RT-qPCR showed that Bna10093360 expressed with less than 0.1% of rapeseed BnaActin,Glyma10g34970.1 and Glyma02g14500.1 expressed with less than 2% of soybean GlymaActin.AhPPC5 expressed 32% to 175% of peanut AhActin,and SIN1018296 expressed 3% to 18% of sesame SINActin.The expression trends of AhPPC5 and SIN1018296 were higher at early seed developing stage and then decreased.Results indicated that the bacterial-type PEPC gene functions were widely differentiated.

Physiological and biochemical responses of the iron chlorosis tolerant grapevine rootstock 140 Ruggeri to iron deficiency and bicarbonate

Iron (Fe) deficiency chlorosis associated with high levels of soil bicarbonate is one of the main nutritional disorders observed in sensitive grapevine genotypes. The aim of the experiment was to assess both the independent and combined effects of Fe and bicarbonate nutrition in grapevine. Plants of the Fe chlorosis tolerant 140 Ruggeri rootstock were grown with and without Fe(III)-EDTA and bicarbonate in the nutrient solution. SPAD index, plant growth, root enzyme (PEPC, MDH, CS, NADP+ −IDH) activities, kinetic properties of root PEPC, organic acid concentrations in roots and xylem sap and xylem sap pH were determined. A factorial statistical design with two factors (Fe and BIC) and two levels of each factor was adopted: +Fe and −Fe, and +BIC and −BIC. This rootstock strongly reacted to Fe deficiency by activating several response mechanisms at different physiological levels. The presence of bicarbonate in the nutrient solution changed the activity of PEPC and TCA related enzymes (CS, NADP+-IDH) and the accumulation/translocation of organic acids in roots of Fe-deprived plants. Moreover, this genotype increased root biomass and root malic acid concentration in response to high bicarbonate levels in the substrate. Bicarbonate also enhanced leaf chlorophyll content. Along with a clear independent effect on Fe nutrition, our data support a modulating role of bicarbonate on Fe deficiency response mechanisms at root level.

Functional Analysis of the Phosphoenolpyruvate Carboxylase on the Lipid Accumulation of Peanut (Arachis hypogaea L.) Seeds

Phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) catalyses phosphoenolpyruvate (PEP) to yield oxaloacetate, which is involved in protein biosynthesis. Pyruvate kinase (PK; EC 2.7.1.40) catalyzes PEP to yield pyruvate, which is involved in fatty acid synthesis. In this study, five PEPC genes (AhPEPC1, AhPEPC2, AhPEPC3, AhPEPC4, and AhPEPC5) from peanut have been cloned. Using a quantitative real-time RT-PCR approach, the expression pattern of each gene was monitored during the seed development of four peanut varieties (E11, Hebeigaoyou, Naihan 1, and Huayu 26). It was found that these five genes shared similar expression behaviors over the developmental stages of E11 with high expression levels at 30 and 40 d after pegging (DAP); whereas these five genes showed irregular expression patterns during the seed development of Hebeigaoyou. In Naihan 1 and Huayu 26, the expression levels of the five genes remained relatively high in the first stage. The PEPC activity was monitored during the seed development of four peanut varieties and seed oil content was also characterized during whole period of seed development. The PEPC activity followed the oil accumulation pattern during the early stages of development but they showed a significantly negative correlation thereafter. These results suggested that PEPC may play an important role in lipid accumulation during the seed development of four peanut varieties tested.

Effects of Fe Deficiency on Organic Acid Metabolism in Pisum sativum Roots

Iron deficiency induces several responses to iron shortage in plants. Metabolic changes occur to sustain the increased iron uptake capacity of Fe-deficient plants. The aim of this work was to investigate the impact of Fe deficiency on the organic acid metabolism in Pisum sativum roots. For this purpose, seedlings of Pisum sativum (cv. Douce) were grown under controlled conditions, in the presence of iron sufficient (C) or deficient (D) mediums. Our results showed that PEPC activity increased by 290% in root extracts of Fe deficient plants, compared to the control. Citrate concentration increased in Fe deficient Pisum roots (114% of the control). As well, MDH, CS and ICDH activities showed a marked increase in roots subjected to D treatment. However, the extent of stimulation was especially important for MDH and ICDH activities (99% and 150% of the control, respectively). These data suggest that the capacity of Douce cultivar to increase organic acid metabolism enzyme activities under iron deficiency is related to its better Fe-use efficiency, which indicate the tolerance level of this cultivar to iron chlorosis.

Capacity for NADPH regeneration in the leaves of two poplar genotypes differing in ozone sensitivity

Cell capacity for cytosolic NADPH regeneration by NADP-dehydrogenases was investigated in the leaves of two hybrid poplar (Populus deltoides × Populus nigra) genotypes in response to ozone (O3 ) treatment (120 ppb for 17 days). Two genotypes with differential O3 sensitivity were selected, based on visual symptoms and fallen leaves: Robusta (sensitive) and Carpaccio (tolerant). The estimated O3 flux (POD0 ), that entered the leaves, was similar for the two genotypes throughout the treatment. In response to that foliar O3 flux, CO2 assimilation was inhibited to the same extent for the two genotypes, which could be explained by a decrease in Rubisco (EC 4.1.1.39) activity. Conversely, an increase in PEPC (EC 4.1.1.31) activity was observed, together with the activation of certain cytosolic NADP-dehydrogenases above their constitutive level, i.e. NADP-G6PDH (EC 1.1.1.49), NADP-ME (malic enzyme) (EC 1.1.1.40) and NADP-ICDH (NADP-isocitrate dehydrogenase) (EC1.1.1.42). However, the activity of non-phosphorylating NADP-GAPDH (EC 1.2.1.9) remained unchanged. From the 11th fumigation day, NADP-G6PDH and NADP-ME profiles made it possible to differentiate between the two genotypes, with a higher activity in Carpaccio than in Robusta. At the same time, Carpaccio was able to maintain high levels of NADPH in the cells, while NADPH levels decreased in Robusta O3 -treated leaves. All these results support the hypothesis that the capacity for cells to regenerate the reducing power, especially the cytosolic NADPH pool, contributes to improve tolerance to high ozone exposure.

Photosynthetic responses of a C3 and three C4 species of the genus Panicum (s.l.) with different metabolic subtypes to drought stress

Young plants of Panicum bisulcatum (C(3)), Zuloagaea bulbosa [NADP-malic enzyme (ME)-C(4)], P. miliaceum (NAD-ME-C(4)) and Urochloa maxima [phosphoenolpyruvate carboxykinase (PCK)-C(4)] were subjected to drought stress (DS) in soil for 6 days. The C(3) species showed severe wilting symptoms at higher soil water potential (-1.1 MPa) and relative leaf water content (77 %) than in the case of the C(4) species (-1.5 to -1.7 MPa; 58-64 %). DS decreased photosynthesis, both under atmospheric and under saturating CO(2). Stomatal limitation of net photosynthesis (P(N)) in the C(3), but not in the C(4) species was indicated by P(N)/C(o) curves. Chlorophyll fluorescence of photosystem II, resulting from different cell types in the four species, indicated NADPH accumulation and non-stomatal limitation of photosynthesis in all four species, even under high CO(2). In the NAD-ME-C(4) and the PCK-C(4) species, DS plants showed increased violaxanthin de-epoxidase rates. Biochemical analyses of carboxylating enzymes and in vitro enzyme activities of the C(4) enzymes identified the most likely non-stomatal limiting steps of photosynthesis. In P. bisulcatum, declining RubisCO content and activity would explain the findings. In Z. bulbosa, all photosynthesis enzymes declined significantly; photosynthesis is probably limited by the turnover rate of the PEPC reaction. In P. miliaceum, all enzyme levels remained fairly constant under DS, but photosynthesis can be limited by feedback inhibition of the Calvin cycle, resulting in asp inhibition of PEPC. In U. maxima, declines of in vivo PEPC activity and feedback inhibition of the Calvin cycle are the main candidates for non-stomatal limitation of photosynthesis under DS.

Fruit Photosynthesis and Phosphoenolpyruvate Carboxylase Activity as Affected by Lightproof Fruit Bagging in Satsuma Mandarin

To clarify why fruit bagging reduces sugar content at harvest, we investigated its effect on carbon dioxide assimilation by Satsuma mandarin (Citrus unshiu) fruit through photosynthesis and phosphoenolpyruvate carboxylase (PEPC; enzyme code 4.1.1.31). Seasonal changes in gross photosynthesis ranged from 70 to 400 μmol·d−2·h−1 O2 with a peak at 99 days after full bloom (DAFB) when the assimilation rate of fruit was comparable to that of leaves. However, a peak showing net photosynthesis appeared at 112 DAFB because of high fruit respiration. When fruit were bagged at 85 DAFB, the net photosynthetic peak disappeared, perhaps as a result of the decline in chlorophyll content in the rind. Sugar and organic acid content in the bagged fruit were 0.3% and 0.16% less, respectively, than controls at the mature stage (204 DAFB). PEPC activity in the rind was much higher than in leaves on a protein basis; it increased between 92 and 112 DAFB and showed a peak of 72 units. The PEPC activity peak was also 90% of control after fruit bagging. Thus, just before their color development, mandarin fruit assimilate CO2 actively through photosynthesis and PEPC. However, these activities are inhibited by bagging, likely resulting in lower sugar content at harvest. The concomitant activation of PEPC and photosynthesis between 99 and 126 DAFB indicates that CO2 fixed by PEPC might be used for photosynthesis in mandarin fruit, because photosynthesis in several fruit such as apple (Malus pumila) and pea (Pisum sativum) is considered to have an intermediate status among C3, non-autotrophic tissue, and C4/CAM photosynthesis.

Responses of two lines of Medicago ciliaris to Fe deficiency under saline conditions

The aim of this research was to study the responses of two lines of Medicago ciliaris: TN11.11 and TN8.7 to iron deficiency under saline conditions. However; the paper showed also the results of a preliminary study which report the contrastive responses of the two lines to salinity. We found that plant growth and chlorophyll content of TN11.11 line were more affected by salinity than TN8.7. The severity of symptoms was linked to the sodium accumulation in shoots as well as a limitation of potassium uptake. Our data allowed us to note that TN8.7 line is less sensitive and can better cope with the salinity. Concerning the effect of salinity on iron deficiency responses, we noted that root PM H+-ATPase and FCR activities were reduced when iron deficiency was associated with salinity. This probably explained the decrease of Fe uptake. On the contrary, PEPC activity was not affected.