Phosphoenolpyruvate Carboxylase Enzyme Activities Research Articles

Comparing the Effects of N and P Deficiency on Physiology and Growth for Fast- and Slow-Growing Provenances of Fraxinus mandshurica

With the continuous increase in atmospheric carbon dioxide emissions, nitrogen (N) and phosphorus (P) as mineral elements increasingly restrict plant growth. To explore the effect of deficiency of P and N on growth and physiology, Fraxinus mandshurica (hereafter “F. mandshurica”) Rupr. annual seedlings of Wuchang (WC) provenance with fast growth and Dailing (DL) provenance with slow growth were treated with complete nutrition or starvation of N (N-), P (P-) or both elements (NP-). Although P- and N- increased the use efficiency of P (PUE) and N (NUE), respectively, they reduced the leaf area, chlorophyll content and activities of N assimilation enzymes (NR, GS, GOGAT), which decreased the dry weight and P or N amount. The free amino acid content and activities of Phosphoenolpyruvate carboxylase (PEPC) and acid phosphatase enzymes were reduced by N-. The transcript levels of NRT2.1, NRT2.4, NRT2.5, NRT2.7, AVT1, AAP3, NIA2, PHT1-3, PHT1-4 and PHT2-1 in roots were increased, but those of NRT2.1, NRT2.4, NRT2.5, PHT1-3, PHT1-4, PHT2-1 and AAP3 in leaves were reduced by P-. WC was significantly greater than DL under P- in dry weight, C amount, N amount, leaf area, PUE, NUE, which related to greater chlorophyll content, PEPC enzyme activity, N assimilation enzyme activities, and transcript levels of N and P transporter genes in roots and foliage, indicating a greater ability of WC to absorb, transport and utilize N and P under P-. WC was also greater than DL under N- in terms of the above indicators except the transcript levels of N and P assimilation genes, but most of the indicators did not reach a significant level, indicating that WC might be more tolerant to N- than DL, which requires further verification. In summary, WC was identified as a P-efficient provenance, as the growth rate was greater for the genetic type with high than low tolerance to P-.

Development of a compartmentalized model for insight into the structured metabolic pathway of carbon metabolism in cassava leaves

In silico metabolic modeling has enabled systematic study of complicated metabolic processes underlying phenotypes of organisms. Modeling of plant metabolism is often hampered by the network complexity and lack of adequate knowledge. The existing metabolic networks of cassava only cover broad metabolism and are not compartmentalized to truly represent metabolism in photosynthetic tissues. To address the aforementioned limitations and develop a robust metabolic network, physiological and genomic data derived from cassava and leaf models of Arabidopsis and rice were to extend the scope of the existing model. The proposed compartmentalized network of metabolism in photosynthetic tissues of cassava, ph-MeRecon (photosynthetic-Manihot esculenta Metabolic Pathway Reconstruction) was developed based on the information resulting of the comparative study of multiple model plants and cassava genome. The ph-MeRecon covers primary carbon metabolism and comprises 461 metabolites, 550 reactions, and 1,037 metabolic genes. Enzymatic genes on the network were validated using RNA-expression data, and the reactions and pathways were compartmentalized into cytoplasm, chloroplast, mitochondria, and peroxisome. To ensure network connectivity, metabolic gaps were filled using gap reactions obtained from literature and metabolic pathway omnibus. In addition, information on plant physiology, including photosynthetic light-dependent reactions, carboxylase and oxygenase activity of RuBisCO enzyme, and phosphoenolpyruvate carboxylase enzyme activity was incorporated into ph-MeRecon to mimic cellular metabolism in cassava leaves. Thus, ph-MeRecon offers a multi-level platform for system analysis of cellular mechanisms underlying phenotypes of interest in cassava. The ph-MeRecon metabolic model is available at http://bml.sbi.kmutt.ac.th/ph-MeRecon/.

Transformation and characterization of transgenic rice and Cleome spinosa plants carrying the maize phosphoenolpyruvate carboxylase genomic DNA

Phosphoenolpyruvate carboxylase (PEPC) is a key enzyme in the C4 photosynthetic pathway for the initial fixation of atmospheric CO2 into the four-carbon (C4) acid, oxaloacetate. Here, we report that a vector carrying the intact maize C4pepc gene was used to transform C3 monocot japonica rice cultivar Tainung 67 (TNG67) which is an important cultivar in Taiwan, and C3 dicot Cleome spinosa which is closely related to the C4 dicot Cleome gynandra, via an Agrobacterium-mediated method. In total, 29 transgenic rice and 4 transgenic C. spinosa plants were obtained. Genomic PCR and southern blot analyses revealed that the maize pepc and selective antibiotic resistant genes were present in almost all randomly-selected transgenic plants but not in wild-type plants. Reverse transcription-PCR showed that maize pepc mRNA was detected in transgenic plants. Western blot analysis confirmed the presence of 110 kDa maize PEPC protein in transgenic plants but not in wild-type plants. Furthermore, higher PEPC enzyme activities were detected in transgenic rice plants than in transgenic C. spinosa plants. Taken together, we clearly demonstrated monocot maize C4pepc gene is functional in both C3 monocot rice and C3 dicot C. spinosa plants. Two T5 homozygous rice lines, each harboring a single insertion of maize pepc gene, showed 7- to 9.4-fold and 40–54% PEPC enzyme activity as compared to the untransformed rice and maize, respectively. These stable transgenic rice lines will be valuable material for studying the effect of maize PEPC on rice photosynthesis under different conditions.

Physiological and photosynthetic characteristics of indica Hang2 expressing the sugarcane PEPC gene

Phosphoenolpyruvate carboxylase (PEPC) is known to play a key role in the initial fixation of CO2 in C4 photosynthesis. The PEPC gene from sugarcane (a C4 plant) was introduced into indica rice (Hang2), a process mediated by Agrobacterium tumefaciens. Integration patterns and copy numbers of the gene was confirmed by DNA blot analysis. RT-PCR and western blotting results showed that the PEPC gene was expressed at both the mRNA and protein levels in the transgenic lines. Real-time PCR results indicated that expression of the sugarcane PEPC gene occurred mostly in green tissues and changed under high temperature and drought stress. All transgenic lines showed higher PEPC enzyme activities compared to the untransformed controls, with the highest activity (11.1 times higher than the controls) being observed in the transgenic line, T34. The transgenic lines also exhibited higher photosynthetic rates. The highest photosynthetic rate was observed in the transgenic line, T54 (22.3 μmol m−2 s−1; 24.6 % higher than that in non-transgenic plants) under high-temperature conditions. Furthermore, the filled grain and total grain numbers for transgenic lines were higher than those for non-transgenic plants, but the grain filling (%) and 1,000-grain weights of all transgenic lines remained unchanged. We concluded that over-expression of the PEPC gene from sugarcane in indica rice (Hang2) resulted in higher PEPC enzyme activities and higher photosynthesis rates under high-temperature conditions.Electronic supplementary materialThe online version of this article (doi:10.1007/s11033-014-3070-4) contains supplementary material, which is available to authorized users.

Thiourea, a ROS Scavenger, Regulates Source-to-Sink Relationship to Enhance Crop Yield and Oil Content in Brassica juncea (L.)

In the present agricultural scenario, the major thrust is to increase crop productivity so as to ensure sustainability. In an earlier study, foliar application of thiourea (TU; a non physiological thiol based ROS scavenger) has been demonstrated to enhance the stress tolerance and yield of different crops under field condition. Towards this endeavor, present work deals with the effect of TU on photosynthetic efficiency and source-to-sink relationship of Indian mustard (Brassica juncea) for understanding its mode of action. The application of TU increased the efficiency of both PSI and PSII photosystems and vegetative growth of plant. The comparative analysis of sucrose to starch ratio and expression level of sugar transporters confirmed the higher source and sink strength in response to TU treatment. The biochemical evidence in support of this was derived from higher activities of sucrose phosphate synthase and fructose-1,6-bis-phosphatase at source; and sucrose synthase and different classes of invertases at both source and sink. This indicated an overall increase in photoassimilate level at sink. An additional contribution through pod photosynthesis was confirmed through the analysis of phosphoenol pyruvate carboxylase enzyme activity and level of organic acids. The increased photoassimilate level was also co-ordinated with acetyl coA carboxylase mediated oil biosynthesis. All these changes were ultimately reflected in the form of 10 and 20% increase in total yield and oil content, respectively under TU treatment as compared to control. Additionally, no change was observed in oil composition of seeds derived from TU treated plants. The study thus signifies the co-ordinated regulation of key steps of photosynthesis and source-to-sink relationship through the external application of TU resulting in increased crop yield and oil content.

Simultaneous Overexpression of Citrate Synthase and Phosphoenolpyruvate Carboxylase in Leaves Augments Citrate Exclusion and Al Resistance in Transgenic Tobacco

Phosphoenolpyruvate carboxylase (PEPC) and citrate synthase (CS) are two key enzymes in organic acid synthesis metabolism. In the present study, a cytoplasmic form of CS from tobacco and a mutant (with reduced sensitivity to organic acid inhibition) PEPC from Synechococcus vulcanus were overexpressed simultaneously using a light-inducible promoter in tobacco leaves. The analysis for enzyme activity showed that CS and PEPC enzyme activities were increased by 235% to 257% and 218% to 236% in the selected cs and pepc (double-gene) overexpression lines, respectively, compared with those in the wild-type plants (WT). The measurement for the relative root elongation rate of the tobacco plants exposed to 30 μM aluminum (Al) indicated that Al tolerance in the double-gene overexpression lines was stronger than that of the transgenic cs or pepc lines and WT plants. The 13C-NMR analysis with NaH13CO3 showed that overexpression of CS and PEPC in the transgenic tobacco successfully constructed a new citrate synthesis pathway. Under the conditions with Al stress, the amount of citrate secreted from the double-transgenic tobacco roots was the largest among the tested plants. When grown on sandy soil supplied with a nutritional solution containing 500 μM Al, the growth of the double-transgenic tobacco was better than that of the transgenic cs or pepc tobacco and WT, and their root biomass was the highest among the tested plants. These results demonstrated that construction of a new citrate synthesis pathway by simultaneous overexpression of CS and PEPC in the cytoplasm of transgenic plant leaves could enhance Al resistance in plants.

Variation for Activity of Nodule Nitrogen and Carbon Assimilating Enzymes in Alfalfa1

The nodule enzymes glutamate synthase (GOGAT) and phosphoenolpyruvate carboxylase (PEPC) are important in the assimilation of symbiotically fixed N in alfalfa (Medicago sativa L.). The objectives of this study were: to evaluate several alfalfa germplasm sources for variability in nodule GOGAT and PEPC activities, and to determine the relationships between N2 fixation and plant yield traits with nodule GOGAT and PEPC enzyme activities. Approximately 160 plants from each of six alfalfa germplasms were grown in sand culture in the glasshouse and assayed for nodule GOGAT and PEPC enzyme activities. Twenty‐four genotypes (four from each germplasm) were selected to represent a range in GOGAT and PEPC activities. These genotypes were evaluated in glasshouse and field clonal studies for GOGAT and PEPC activities per gram nodule fresh weight, acetylene reduction activity (ARA), nodule mass, nodule color, nodule soluble protein concentration, and plant yield. Variability for nodule GOGAT and PEPC activities within each of the six germplasms was large and normally distributed. The 12 high GOGAT genotypes (2 from each germplasm) were 44% greater GOGAT activity than the 12 low genotypes. The 12 high PEPC genotypes were 27% greater in PEPC activity than the 12 low genotypes. Both groups of high enzyme activity genotypes were significantly greater than the low genotypes in ARA, nodule soluble protein concentration, and field shoot yield. Both GOGAT and PEPC activities were positively correlated (r=0.61**, significant at the 0.01 probability level). Also, GOGAT and PEPC were each correlated with ARA (r=0.43*, significant at the 0.05 probability level, and 0.45*, respectively), nodule color score (r=0.46** and 0.73**) soluble protein concentration (r= 0.77** and 0.90**). Plant yield and N2 fixation measured in the field were not correlated with nodule GOGAT and PEPC activities measured in the glasshouse. The relationships between GOGAT and PEPC with other plant traits indicated that these enzymes are associated with the host‐Rhizobium meliloti Dang. symbiosis and should be explored as possible selection criteria to include in an alfalfa breeding program.

Relationship between leaf development, carboxylase enzyme activities and photorespiration in the C4-plant Portulaca oleracea L.

Ribulose diphosphate (RuDP) and (PEP) phosphoenolpyruvate carboxylase enzyme activities were studied in young, mature, and senescent Portulaca oleracea leaves. While the absolute amount of both the C3 (RuDP) and C4 (PEP) carboxylase is less in senescent leaves than in mature leaves, RuDP carboxylase activity is reduced to a lesser degree. In senescent leaves, PEP carboxylase activity equals 10% of that in mature tissue, but RuDP carboxylase is 27% of that in mature leaves. The same ontogenetic series was also used to determine photorespiration rates and responses to several gas treatments. Young and mature leaves were unaffected by changes in the light regime or oxygen concentrations, and exhibited typical C4-plant light/dark (14)CO2 evolution ratios. Senescent leaves, on the other hand, have photorespiration ratios similar to C3-plants. In addition, senescent leaves were affected by minus CO2, 100% O2 and N2 in a manner expected of C3-plants, but not C4-plants. These results are discussed in terms of a relative increase in activity of the C3 cycle in later developmental stages in this plant.