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.     

Accumulation of Carbohydrate and Regulation of 14-3-3 Protein on Sucrose Phosphate Synthase(SPS) Activity in Two Tomato Species

To explore the differences of carbohydrate metabolism in two tomato species and discuss the possible regulation of 14-3-3proteins on the sucrose phosphate synthase(SPS)activity,we determined the contents of soluble sugar and starch through high performance liquid chromatography(HPLC).The activities of sugar-metabolizing enzymes were assayed in desalted extract,and the relative expression levels of related genes in sugar metabolism were determined though real-time RT-PCR.The results indicated that glucose and fructose were mainly accumulated during the maturation of the fruit because of the high acid invertase(AI)and neutral invertase(NI)in Micro-Tom(Solanum lycopersicum)fruit,while in Solanum chmielewskii fruit,SPS which went along with the change of sucrose content led to the rapid sucrose increase during the fruit ripening.TFT1and TFT10,belonging to 14-3-3 protein in tomato,were likely to down-regulated SPS activity during young and intumescence period.     

Leaf Photosynthesis in Response to Removing Fruit During Different Phenological Stages of Fruit Development in Peach Trees

Removing fruit (RF) and retaining fruit (CK) were carried out during different phenological stages of fruit development on one-year-old shoot of Okubo peach trees [Prunus persica (L.) Batsch.] under preventing exportation of the assimilates to the non experimental parts of the tree by girdling one-year-old shoot and keeping the same leaves between RF and CK. The results showed that fruit removal significantly decreased net photosynthetic rate (Pn), stomatal conductance (Gs) and transpiration rate (E), but significantly increased leaf surface temperature (TLeaf) at about midday as compared with CK. Internal CO2 concentration, soluble sugar content, reductive sugar content, starch content except that during the final rapid fruit growth stage, ADP-glucose pyrophosphorylase and amylase activities in source leaves were not significantly affected by fruit removal. There was a significantly positive parabolic correlation between Pn and Gs, and a strong positive linear correlation between Pn and E. Moreover, Pn increased with increased TLeaf if TLeaf was below 38℃, thendecreased sharply when TLeaf exceeded the above critical temperature for both RF and CK. Pn of RF was lower, however, than that of CK in the same TLeaf, especially if TLeaf exceeded 38℃. It is suggested that the decreased stomatal aperture and increased TLeaf may be the important mechanism in regulating photosynthesis under a decreased strength of sink demand by RF in fruit trees.     

Genome-wide identification,molecular evolution,and expression divergence of the hexokinase gene family in apple

Hexokinase(HXK) is the first irreversible catalytic enzyme in the glycolytic pathway, which not only provides energy for plant growth and development but also serves as a signaling molecule in response to environmental changes. However, the evolutionary pattern of the HXK gene family in apple remains unknown. In this study, a total of nine HXK genes were identified in the Malus×domestica genome GDDH13 v1.1. The physiological and biochemical properties, exonintron structures, conserved motifs, and cis-elements of the MdHXK genes were determined. Predicted subcellular localization indicated that the MdHXK genes were mainly distributed in the mitochondria, cytoplasm, and nucleus. Gene duplication revealed that whole-genome duplication(WGD) and segmental duplication played vital roles in MdHXK gene family expansion. The ω values of pairwise MdHXK genes indicated that this family was subjected to strong purifying selection during apple domestication. Additionally, five subfamilies were classified, and recent/old duplication events were identified based on phylogenetic tree analysis. Different evolutionary rates were estimated among the various HXK subfamilies. Moreover, divergent expression patterns of the Md HXK genes in four source-sink tissues and at five different apple fruit developmental stages indicated that they play vital roles in apple fruit development and sugar accumulation. Our study provides a theoretical basis for future elucidation of the biological functions of the MdHXK genes during apple fruit development.     

Expression of an Episomal Vector in Developing Chicken Embryo and Chicks

The domesticated chicken has important roles in basic and applied research. The vector based on scaffold matrix attachment region （S/MAR） appears to be sufficient to maintain long-term expression in an episomal state in various mammalian cells. To explore the practical use of episomal vector in transgene technology of agricultural chickens, we fused the S/MAR of the human r-interferon gene into the pEGFP vector and transduced it into chickens by sperm-mediated gene transfer （SMGT）. PCR detection indicated the positive rate of transgene chickens was 60%. The RT-PCR detection and fluorescence observation confirmed the expression of the GFP and indicated the existence of the GFP during the chicken embryogenesis and fetal development. The PCR detection and rescue experiments confirmed the episomal state of the pEPI-EGFP in chick embryos and chicks. These results showed that the S/MAR-based vector could function properly in chicken embryos and was a practicable tool combined with the SMGT to study the development of chickens.     

Two Lycopene β-Cyclases Genes from Sweet Orange （Citrus sinensis L. Osbeck） Encode Enzymes With Different Functional Efficiency During the Conversion of Lycopene-to-Provitamin A

Citrus fruits are rich in carotenoids.In the carotenoid biosynthetic pathway,lycopene β-cyclase（LCYb,EC：1.14.-.-） is a key regulatory enzyme in the catalysis of lycopene to β-carotene,an important dietary precursor of vitamin A for human nutrition.Two closely related lycopene β-cyclase cDNAs,designated CsLCYb1 and CsLCYb2,were isolated from the pulp of orange fruits（Citrus sinensis）.The expression level of CsLCYb genes is lower in the flavedo and juice sacs of a lycopeneaccumulating genotype Cara Cara than that in common genotype Washington,and this might be correlated with lycopene accumulation in Cara Cara fruit.The CsLCYb1 efficiently converted lycopene into the bicyclic β-carotene in an Escherichia coli expression system,but the CsLCYb2 exhibited a lower enzyme activity and converted lycopene into the β-carotene and the monocyclic γ-carotene.In tomato transformation studies,expression of CsLCYb1 under the control of the cauliflower mosaic virus（CaMV） 35S constitutive promoter resulted in a virtually complete conversion of lycopene into β-carotene,and the ripe fruits displayed a bright orange colour.However,the CsLCYb2 transgenic tomato plants did not show an altered fruit colour during development and maturation.In fruits of the CsLCYb1 transgenic plants,most of the lycopene was converted into β-carotene with provitamin A levels reaching about 700 μg g-1DW.Unexpectedly,most transgenic tomatoes showed a reduction in total carotenoid accumulation,and this is consistent with the decrease in expression of endogenous carotenogenic genes in transgenic fruits.Collectively,these results suggested that the cloned CsLCYb1 and CsLCYb2 genes encoded two functional lycopene β-cyclases with different catalytic efficiency,and they may have potential for metabolite engineering toward altering pigmentation and enhancing nutritional value of food crops.     

Identification and expression of the CEP gene family in apple (Malus×domestica)

Plant peptide hormones play important roles in plant growth and development. Among these hormones, the C-TERMINALLYENCODED PEPTIDE(CEP) belongs to a newly found peptide family that regulates root development in Arabidopsis as well as in other species. However, nothing is known about the CEP genes in apple(Malus×domestica, MdCEP). In this study, a total of 27 apple CEP genes were identified through a genome-wide analysis and were phylogenetically divided into three classes(Ⅰ, Ⅱ and Ⅲ). The predicted MdCEP genes were distributed across 10 of 17 chromosomes with different densities. Next, the gene structures and motif compositions of the MdCEP genes were analyzed. Subsequently, the expression analysis suggested that the MdCEP genes were highly activated in roots and that MdCEP23 may play an important role in regulating the growth and development of roots. Moreover, all of the MdCEP genes were responsive to multiple abiotic stresses, indicating that MdCEP genes may be involved with various aspects of physiological processes in apple. Nearly one-third of MdCEP genes had a significant response to low nitrogen treatment. Most of the MdCEP genes were up-regulated under stress, including mannitol, polyethylene glycol(PEG) and abscisic acid(ABA), suggesting that MdCEP genes may be involved in the drought stress response. This study provides insight into the putative functions of the MdCEP genes using a genome-wide analysis of the CEP gene family.     

Accumulation of Carbohydrate and Regulation of 14-3-3 Protein on Sucrose Phosphate Synthase （SPS）Activity in Two Tomato Species

To explore the differences of carbohydrate metabolism in two tomato species and discuss the possible regulation of 14-3-3 proteins on the sucrose phosphate synthase （SPS） activity, we determined the contents of soluble sugar and starch through high performance liquid chromatography （HPLC）. The activities of sugar-metabolizing enzymes were assayed in desalted extract, and the relative expression levels of related genes in sugar metabolism were determined though real-time RT-PCR. The results indicated that glucose and fructose were mainly accumulated during the maturation of the fruit because of the high acid invertase （AI） and neutral invertase （NI） in Micro-Tom （Solanum lycopersicum） fruit, while in Solanum chmielewskii fruit, SPS which went along with the change of sucrose content led to the rapid sucrose increase during the fruit ripening. TFT1 and TFT10, belonging to 14-3-3 protein in tomato, were likely to down-regulated SPS activity during young and intumescence period.     

EalspF1, Essential Enzyme in Isoprenoid Biosynthesis from Eupatorium adenophorum,Reveals a Novel Role in Light Acclimation

Isoprenoids are a functionally and structurally diverse class of natural organic chemicals. The universal precursors of all isoprenoids, isopentenyl diphosphate and dimethylallyl diphosphate are synthesized through the mevalonate and 2C-methyl- D-erythritol 4-phosphate （MEP） pathways, respectively. Many isoprenoids produced through the MEP pathway play an important role in plant acclimation to different light environments. Eupatorium adenophorum, an invasive weed in China, presents a remarkable capacity to acclimate to various light environments, which constitutes its solid foundation of being a successful invasive species. Thus we aimed at gaining a deeper insight into the regulation of MEP pathway in E. adenophorum to further understand the invasive mechanism. 2C-Methyl-D-erythritol 2,4-cyclodiphosphate synthase （IspF or MCS） is an essential enzyme in the MEP pathway. In this paper, a novel IspF gene was cloned and characterized from E. adenophorum. Tissue-specific expression assays revealed a higher expression of EalspF1 in leaves than in stems and roots. The expression of EalspF1 was responsive to different light conditions. Some up-regulation of EalspF1 expression was also found after the treatments with signal compounds and after wounding stress. Interestingly, the over-expression of EalspF1 in Arabidopsis led to increase carotenoids contents, resulting in an enhanced tolerance to high light. Taken together, these results indicate that the EalspFl-derived enzyme participates in isoprenoid metabolism and among others, the expression of this gene in E. adenophorum is involved in the regulation of plastidial isoprenoids, which play an important role in acclimation to various light environments.     

Reduction in Activity/Gene Expression of Anthocyanin Degradation Enzymes in Lychee Pericarp is Responsible for the Color Protection of the Fruit by Heat and Acid Treatment

Heat and acid treatments were reported to be a promising substitute for SO₂ fumigation in color protection of postharvest lychee （Litchi chinensis Sonn.） fruits, but the mechanism was not clear. In the present study, hot water （70°C） dipping followed by immersion in 2% HCl （heat-acid） substantially protected the red color of the fruit during storage at 25°C and inhibited anthocyanin degradation while hot water dipping alone （heat） led to rapidly browning and about 90% loss in anthocyanin content. The pH values in the pericarp of the heat-acid treated fruit dropped to 3.2, while the values maintained around 5.0 in the heat-treated and control fruit. No significantly different pH values were detected among the arils of heat-acid, heat treated and control fruit. Heat-acid treatment dramatically reduced the activities of anthocyanin degradation enzyme （ADE）, peroxidase （POD） and polyphenol oxidase in the pericarp. A marked reduction in LcPOD gene expression was also detected in heat-acid treated fruit, in contrast, induction was found in heat treated fruit. The pericarp of heat-acid treated fruit exhibited significantly lower respiration rate but faster water loss than that of the untreated or heat treated fruit. Taken together, heat treatment triggered quick browning and anthocyanin loss in lychee fruit, while heat-acid treatment protected the fruit color by a great reduction in the activities/gene expression of anthocyanin degradation enzymes and acidification of lychee perica.     

Hoxc13 Expression Pattern in Cashmere Goat Skin During Hair Follicle Development

Hoxc13 has an important role in controlling hair formation. In this study, we examine the Hoxc13 RNA expression pattern of skin during embryo development. The result indicated that changes of the Hoxe13 gene expression and thickness of skin have a similar trend during hair follicle morphogenesis. In interpreting these results, we investigated whether the regulation motifs is in Hoxc13 intron, which is a 5.4 kb fragment. To blast with other mammals, we found a very conservative region in all mammal animals and two regions in livestock, such as cow, sheep, horse, dog, and so on, which are not in other Hox genes. We have examined putative pre-miRNA in this region, providing an entry point for elucidating currently unknown mechanisms that are required for regulating quantitative levels of Hoxc13 gene expression.     

In Silico Cloning and Sequence Analysis of Phospholipase Dα Gene from Peach Fruit

Phospholipase D （PLD, EC 3.1.4.4） plays an important role in adaptive response of postharvest fruit to environment. In this study, a novel cDNA of PLDα was isolated with the strategy of in silico cloning in combination with RT-PCR from peach （Prunus persica L. cv. Jiubao）. The obtained PLDα gene contained a complete open reading frame encoding a 92- kDa protein of 810 amino acid residues, which possessed the characteristic C2 domain and two catalytic HKD motifs. The alignment analysis of the deduced peach PLDa protein with other known PLDα family proteins indicated that peach PLDα was conserved and highly homologous with strawberry PLDα. Semi-quantitative RT-PCR and Northern blot analysis indicated PLDα mRNA in peach fruits could be induced by low temperature. This work provided a scientific basis for further investigating the mechanism of postharvest fruit adaptation to low temperature.