Preliminary Study on Phosphoenolpyruvate Carboxylase(PEPCase) Gene Introduced into Wheat

[ Objective] The aim of this study was to introduce Phosphoenolpyruvate Carboxylase （PEPCase） gene into common wheat Linyou 145. [ Method] With the material of common wheat Linyou 145, Phosphoenolpyruvate Carboxylase （PEPCase） gene was introduced into wheat embryo callus by the agrobacterium-mediated transformation system, and then analyzed through successive selection with selective medium con- taing gygrornycin to detect the gene at the molecular level. [Result] The hyg-resistant plants were obtained, and GUS histochemical staining showed the leaf of resistant plants was stained dark blue. The target bands appeared in PCR analysis. [ Conclusion] Phosphoenolpyruvate Car- boxylase （PEPCase） gene has been primarily introduced into the recipient material.

Identification and Expression Analysis of the Phosphoenolpyruvate Carboxylase Gene Family in Peanut (Arachis hypogaea L.)

Phosphoenolpyruvate carboxylase （PEPC） is widely distributed in plants and bacteria, and catalyzes the carboxylation of phosphoenolpyruvate to form oxaloacetate and inorganic phosphate. To investigate the molecular mechanisms of the regulation and control of peanut oil, with the degenerated primers and RACE-PCR approach, five PEPC genes were cloned from peanut, and designated as AhPEPC1, AhPEPC2, AhPEPC3, AhPEPC4, and AhPEPC5, respectively. The structure and phylogenetic analysis of PEPC protein indicated that AhPEPC1-4 genes encoded a typical plant-type PEPC-enzyme, and AhPEPC5 a bacterial-type. By real-time quantitative RT-PCR approach the expression pattern of each gene was detected in various tissues of normal and high oil-content peanut varieties. It was found that there was a lower expression level of AhPEPCs genes except for the AhPEPC2 in high-oil peanut than normal-oil peanut line. The results provide some fundamental information for the further investigation of plant PEPC proteins and their role in regulation of oil-content in peanut seeds.

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.

Photosynthetic Characteristics and Heterosis in Transgenic Hybrid Rice with Maize Phosphoenolpyruvate Carboxylase (pepc) Gene

Three F3 hybrids derived from the sterile rice lines Gang 46A, 776A and 2480A and the improved restorer line Shuhui 881 containing maize phosphoenolpyruvate carboxylase （pepc） gene were used to analyze the effect of pepc gene on the heterosis and photosynthetic characteristics, while the F3 obtained by crossing Shuhui 881 with the above three sterile lines served as controls. The dynamics of photosynthetic characteristics in leaves of three F1 with pepc gene and their controls were determined at the initial-tillering, maxium-tillering, elongation, initial-heading, heading, maturity stages, and other different times after flag leaf fully expanded. The PEPCase activities of the three F1 with pepc gene increased significantly as compared with control plants during the whole developmental stages. Moreover, the net photosynthesis rate （Pn） also increased to certain extent. The data showed that PEPCase activity was significantly correlated to Pn with a correlation coefficient of 0.6081. The photosynthetic indexes of the three F1 with pepc gene were obviously superior to respective controls in apparent quantum efficiency, light compensation point and carboxylation efficiency, while the CO2 compensation point was lower than that of corresponding control. The Pn of the three F1 with pepc gene at light saturation point and CO2 saturation point was also higher than that of control plants. in addition, the three F1 with pepc gene had an average increase of 37.10% in grain yields per plant in comparison with control plants. The results indicated that the photosynthetic characteristics of hybrid rice containing pepc gene had been improved to some extent due to the introduction of pepc gene.

Promotive Effect of Low Concentrations of NaHSO3 on Photophosphorylation and Photosynthesis in Phosphoenolpyruvate Carboxylase Transgenic Rice Leaves

: Spraying a 1–2 mmol/L solution of NaHSO3 on the leaves of wild-type rice (Oryza sativa L.) Kitaake (WT), phosphoenolpyruvate carboxylase (PEPC) transgenic (PC) rice and PEPC+phosphate dikinase (PPDK) transgenic rice (PC+PK), in which the germplasm was transformed with wild-type Kitaake as the gene receptor, resulted in an enhancement of the net photosynthetic rate by 23.0%, 28.8%, and 34.4%, respectively, for more than 3 d. It was also observed that NaHSO3 application caused an increase in the ATP content in leaves. Spraying PMS (a cofactor catalysing the photophosphorylation cycle) and NaHSO3 separately or together on leaves resulted in an increase in photosynthesis with all treatments. There was no additional effect on photosynthetic rate when the mixture was applied, suggesting that the mechanism by which NaHSO3 promotes photosynthesis is similar to the mechanism by which PMS acts and that both of compounds enhanced the supply of ATP. After spraying a solution of NaHSO3 on leaves, compared with the WT Kitaake rice, a greater enhancement of net photosynthetic rate was observed in PEPC transgenic (PC) and PEPC+PPDK transgenic (PC+PK) rice, with the greatest increase being observed in the latter group. Therefore ATP supply may become the limiting factor that concentrates CO2 in rice leaves transformed with an exogenous PEPC gene and exogenous PEPC+PPDK genes.

CO2 Exchange and Chlorophyll Fluorescence of Phosphoenolpyruvate Carboxylase Transgenic Rice Pollen Lines

To elucidate the photosynthetic physiological characteristics and the physiological inherited traits of rice （Oryza sativa L.） hybrids and their parents, physiological indices of photosynthetic CO2 exchange and chlorophyll fluorescence parameters were measured in leaves of the maize phosphoenolpyruvate carboxylase （PEPC） transgenic rice as the male parent, sp. japonica rice cv. 9516 as the female parent, and the stable JAAS45 pollen line. The results revealed that the PEPC gene could be stably inherited and trans- ferred from the male parent to the JAAS45 pollen line. Moreover, the JAAS45 pollen line exhibited high levels of PEPC activity, manifesting higher saturated photosynthetic rates, photosynthetic apparent quantum yield （AQY）, photochemical efficiency of photosystem II and photochemical and non-photochemical quenching, which indicated that the JAAS45 pollen line has a high tolerance to photo-inhibition/photooxidation under strong light and high temperature. Furthermore, JAAS45 was confirmed to still be a C3 plant by δ^13C carbon isotope determination and was demonstrated to have a limited photosynthetic C4 microcycle by feeding with exogenous C4 primary products, such as oxaloacetate or malate, or phosphoenolpyruvate. The present study explains the physiological inherited properties of PEPC transgenic rice and provides an expectation for the integration of traditional breeding and biological technology.

Plasticity of photorespiratory carbon concentration mechanism in Sedobassia sedoides(Pall.)Freitag&G.Kadereit under elevated CO_(2)concentration and salinity

Rising atmospheric CO_(2)(carbon dioxide)concentrations and salinization are manifestations of climate change that affect plant growth and productivity.Species with an intermediate C_(3)-C_(4)type of photosynthesis live in a wide range of precipitation,temperature,and soil quality,but are more often found in warm and dry habitats.One of the intermediate C_(3)-C_(4)photosynthetic type is C_(2)photosynthesis with a carbon concentration mechanism(CCM)that reassimilates CO_(2)released via photorespiration.However,the ecological significance under which C_(2)photosynthesis has advantages over C_(3)and C_(4)plants remains largely unexplored.Salt tolerance and functioning of CCM were studied in plants from two populations(P1 and P2)of Sedobassia sedoides(Pall.)Freitag&G.Kadereit Asch.species with C_(2)photosynthesis exposed to 4 d and 10 d salinity(200 mM NaCl)at ambient(785.7 mg/m^(3),aCO_(2)and elevated(1571.4 mg/m^(3),eCO_(2))CO_(2).On the fourth day of salinity,an increase in Na+content,activity catalase,and superoxide dismutase was observed in both populations.P2 plants showed an increase in proline content and a decrease in photosynthetic enzyme content:rubisco,phosphoenolpyruvate carboxylase(PEPC),and glycine decarboxylase(GDC),which indicated a weakening of C_(2)and C_(4)characteristics under salinity.Treatment under 10 d salinity led to an increased Na^(+)content and activity of cyclic electron flow around photosystem I(PSI CEF),a decreased content of K^(+)and GDC in both populations.P1 plants showed greater salt tolerance,which was assessed by the degree of reduction in photosynthetic enzyme content,PSI CEF activity,and changes in relative growth rate(RGR).Differences between populations were evident under the combination of eCO_(2)and salinity.Under long-term salinity and eCO_(2),more salt-tolerant P1 plants had a higher dry biomass(DW),which was positively correlated with PSI CEF activity.In less salt-tolerant P2 plants,DW correlated with transpiration and dark respiration.Thus,S.sedoides showed a high degree of photosynthetic plasticity under the influence of salinity and eCO_(2)through strengthening(P1 plants)and weakening C_(4)characteristics(P2 plants).

Marker-assisted Selection of ZmC_4Ppc in Rice Breeding and Yield Trait Performances of Advanced Lines

The full-length of intact Zea mays gene for phosphoenolpyruvate carboxylase gene （ZmC4Ppc） is 6 781 bp. The products of PCR for this gene were not clear with poor repeatability, resulting in that it was difficult for marker-assisted selection （MAS） both in rice and maize. For selecting the markers for MAS, sequences presented only in maize rather than in rice were identified by BLAST, and used for primer design using Primer Premier 5.0. A pair of specific primer termed MRpc （Forward： 5＇ AAGCAGGGAAGCGAGACG 3＇, Reverse： 5＇ GATTGCCGCCAGCAGTAG 3＇） was used for selection of transformed rice, and ZmC4Ppc could be highly and constitutively expressed at each tested developmental stages in the transformed rice selected by using MRpc. Thus, MRpc was used for MAS of progenies carrying ZmC4Ppc gene in rice and some restorer lines with ZmC4Ppc （e.g. FPM881） derived from ZmC4Ppc-transformed Kitaake backcrossed with a restorer line Shuhui 881 were obtained. The analyses on genetic background, PEPCase activity, net photosynthetic rate, general combining ability （GCA） and specific combining ability （SCA） of FPM881 showed that similarity of genetic background reached above 95%, the PEPCase and net photosynthetic rate were higher than those of the control, and some of the progenies carrying ZmC4Ppc gene had better GCA and SCA for grain yield per plant, number of panicles per plant, and 1000-grain weight than those of the control. This suggested that the introduction of maize ZmC4Ppc gene via MAS and its stable expression could increase grain yield of rice and would likely provide a pathway for rice varietal improvement.

Improved oxidative tolerance in suspension‐cultured cells of C_4-pepctransgenic rice by H_2O_2 and Ca^(2+)under PEG-6000

To understand the molecular responses of PC （Overexpressing the maize C4‐pepc gene, which encodes phosphoenolpyruvate carboxylase （PEPC））, to drought stress at cel level, we analyzed changes in the levels of signaling molecules （hydrogen peroxide （H2O2）, calcium ion （Ca2t）, and nitric oxide （NO）） in suspension‐cultured PC and wild‐type （WT） rice （Oryza sativa L.） cel under drought stress induced by 20%polyethylene glycol 6000 （PEG‐6000）. Results demonstrated that PC improved drought tolerance by enhancing antioxidant defense, retaining higher relative water content, survival percentages, and dry weight of cel s. In addition, PEPC activity in PC under PEG treatment was strengthened by addition of H2O2 inhibitor, dimethylthiourea （DMTU） and NO synthesis inhibitor, 2‐（4‐carboxyphenyl‐4,4,5,5‐tetramethylimidazoline‐1‐oxyl‐3‐oxide （cPTIO）, respectively, while that in PC was weakened by addition of free calcium chelator, ethylene glycol‐bis（b‐aminoethylether）‐N,N,N0 ,N0‐tetraacetic acid （EGTA） t calcium channel outflow inhibitor, ruthenium red （RR） t plasma membrane channel blocker La（NO3）3, but EGTA t RR did not. Results also showed that NO and Ca2t was lying downstream of H2O2 in drought‐induced signaling. Calcium ion was also involved in the expression of C4‐pepc in PC. These results suggested that PC could improve oxidative tolerance in suspension‐cultured cel s and the acquisition of this tolerance required downregulation of H2O2 and the entry of extracel ular Ca2t into cel s across the plasma membrane for regulation of PEPC activity and C4‐pepc expression.

Dynamic Regulation of Nitrogen and Organic Acid Metabolism of Cherry Tomato Fruit as Affected by Different Nitrogen Forms

Cherry tomatoes （Lycopersicon esculentura Mill., cv. hongyangli） were hydroponically cultivated in a greenhouse to determine the effect of different nitrogen （N） forms on organic acid concentration and the activities of related enzymes involved in nitrogen and organic acid metabolism during cherry tomato fruit development. The results showed that fruit nitrate reductase （NR） activity was much higher following treatment with 100% NO3 and 75% NO3 ＋ 25% NH＋ than with 100% NH＋ except at maturity. Glutamine synthetase （GS） activity trended downward during fruit development under all three treatments. Plants fed 100% NH＋ had the lowest fruit citrate and malate levels at maturity, with the highest malate concentration at an early stage. The activity of phosphoenolpyruvate carboxylase （PEPC） was found to be in accord with the malate concentration with every N source. Under all three N forms, the citrate synthase （CS） activity peaked one week before the citrate concentration.

Transcriptomic changes associated with PCK1 overexpression in hepatocellular carcinoma cells detected by RNA-seq

Phosphoenolpyruvate carboxykinase 1(PCK1),a step limiting enzyme of gluconeogenesis,is downregulated in hepatocellular carcinoma(HCC).Overexpression of PCK1 has been shown to suppress hepatoma cell growth,but the underlying mechanism remains unclear.We used recombinant adenovirus overexpressing PCK1 or GFP in Huh7 cells,and the differentially expressed genes(DEGs)were identified by RNA-Seq.180 were upregulated by PCK1 overexpression,whereas 316 were downregulated.Pathway analysis illustrated that PCK1 was closely correlated with Wnt signaling pathway and TGF-beta signaling pathway.Hence,Wnt signaling pathway and its downstream component,FZD2,FZD6,FZD7 and b-catenin were confirmed by qRT-PCR and Western blot.In vivo we also observed that PCK1 had restrained tumor growth as a result of decreasing expression of b-catenin.Whole-transcriptomic profile analysis discovered that overexpression of PCK1 downregulates several oncogenic signaling pathways in HCC,providing potential therapeutic targets for improving HCC therapy.

Improve gamma‑aminobutyric acid production in Corynebacterium glutamicum by optimizing the metabolic fux

Gamma-aminobutyric acid is an important nonprotein amino acid and has been extensively applied in pharmaceuticals,livestock,food additives,and so on.It is important to develop Corynebacterium glutamicum strains that can efciently produce gamma-aminobutyric acid from glucose.In this study,production of gamma-aminobutyric acid in C.glutamicum CGY700 was improved by construction of CO_(2) anaplerotic reaction and overexpression of citrate synthase.The co-expression of ppc encoding phosphoenolpyruvate carboxylase and gltA encoding citrate synthase was constructed and optimized in the chromosome to compensate carbon loss and conquer metabolic bottleneck.The expression of ppc and gltA were controlled by promoters P_(tac) and P_(tacM),and the optimal mode of P_(tacM-ppc)-P_(tac)-gltA was determined.Simultaneously,the genes pknG encoding serine/threonine protein kinase G and ldh encoding l-lactate dehydrogenase were deleted,and glnA2 encoding glutamine synthase was overexpressed in the chromosome.The fnal strain CGY-PG-304 constructed in this study could produce 41.17 g/L gamma-aminobutyric acid in shake fask cultivation and 58.33 g/L gamma-aminobutyric acid via FedBatch fermentation with a yield of 0.30 g/g glucose.CGY-PG-304 was constructed by genome editing;therefore,it is stable and not necessary to add any antibiotics and inducer during fermentation.