Whole genome identification and analysis of FK506-binding protein family genes in grapevine (Vitis vinifera L.)

In plant and animal species FK506-binding protein (FKBP) family genes are important conserved genes and it is defined as the receptors of FK506 and rapamycin, where they work as PPIase and protein folding chaperones. FKBP have been isolated from Arabidopsis thaliana, Oryza sativa, and Zea mays. In grape, twenty-three genes containing the FK506-binding domain (FKBP_C) were first time identified by HMMER and blast research, they were classified into three groups and 17 out of the 23 genes were located on 11 chromosomes (Chr1, 3, 5, 7, 8, 14, 15, 16, 17, 18, and 19). The predicted gene expression pattern and semi-quantitative RT-PCR results revealed that five VvFKBPs were expressed in all tissues, while seven VvFKBPs were expressed only in some of the tissues, and the remaining VvFKBPs were not expressed in leaf, stem, inflorescences, flowers, and a mixture of fruit tissues (small, medium and big-sized fruits). Most of the VvFKBPs in grapevine ‘Summer Black’ were similar to those predicted one in ‘Pinot Noir’ except for VvFKBP16-4 and VvFKBPa. VvFKBP12, FaFKBP12 and PpFKBP12 were cloned from ‘Summer Black’, ‘Sweet Charlie’ and ‘Xiahui 6’. Protein structure analysis confirmed that homologous genes have some differences during the process of protein structure construction. In this study, we characterized and verified 23 FKBP family genes in grapevine (Vitis vinifera L.) as well as their sub-cellular and chromosome location. The successful cloning of CDS regions and protein structural analysis of VvFKBP12, FaFKBP12, and PpFKBP12 can provide useful information for further study.     

Synthesis and biological evaluation of novel benzyl-substituted (S)-phenylalanine derivatives as potent dipeptidyl peptidase 4 inhibitors

A series of novel benzyl-substituted (S)-phenylalanine derivatives were synthesized and evaluated for their dipeptidyl peptidase 4 (DPP-4) inhibitory activity and selectivity. It was found that most synthesized target compounds were potent DPP-4 inhibitors with IC₅₀ values in 3.79–25.52 nM, which were significantly superior to that of the marketed drug sitagliptin. Furthermore, the 4-fluorobenzyl substituted phenylalanine derivative 6g not only displayed the potent DPP-4 inhibition with an IC₅₀ value of 3.79 nM, but also showed better selectivity against DPP-4 over other related enzymes including DPP-7, DPP-8, and DPP-9. In an oral glucose tolerance test (OGTT) in normal Sprague Dawley rats, compound 6g reduced blood glucose excursion in a dose-dependent manner.     

Changes in soil carbon flux and carbon stock over a rotation of poplar plantations in northwest China

Forest soil is a major component of terrestrial ecosystems for carbon sequestration and plays an important role in the global carbon cycle. Soil carbon flux and soil carbon pools were investigated in a poplar plantation chronosequence over a rotation in northwest China. Based on continuous field observation in 2007, the results showed that mean soil CO₂ efflux rate was 5.54, 4.81, and 3.93 μmol CO₂ m⁻² s⁻¹ for stands of 2-, 8-, and 15-year-old, respectively, during the growing season. Significant differences in soil respiration of three age classes were mainly because soil temperature, carbon allocation, and fine root growth changed greatly with stand age. Multiple regression analysis suggested that soil temperature and fine root biomass in the upper layer could explain 78–85% of the variation in soil respiration. Mineral soil C stock at 0–40 cm depth was 55.77, 55.09, and 58.14 t ha⁻¹ in the 2-, 8-, and 15-year-old stands, respectively. The average rate of soil C sequestration was 0.13 t ha⁻¹ year⁻¹ following afforestation on former crop lands. Although the plantations had similar management practices and soil types since their establishment, many biotic and abiotic factors such as root biomass and turnover rate, soil condition of the plantations had undergone marked changes at different development stages, which could result in the remarkable differences in soil carbon flux and storage over a rotation. Our results highlight the importance of the development stage within a rotation of poplar plantation in assessment of soil carbon budget.     

Genetic control of a transition from black to straw-white seed hull in rice domestication

The genetic mechanism involved in a transition from the black-colored seed hull of the ancestral wild rice (Oryza rufipogon and Oryza nivara) to the straw-white seed hull of cultivated rice (Oryza sativa) during grain ripening remains unknown. We report that the black hull of O. rufipogon was controlled by the Black hull4 (Bh4) gene, which was fine-mapped to an 8.8-kb region on rice chromosome 4 using a cross between O. rufipogon W1943 (black hull) and O. sativa indica cv Guangluai 4 (straw-white hull). Bh4 encodes an amino acid transporter. A 22-bp deletion within exon 3 of the bh4 variant disrupted the Bh4 function, leading to the straw-white hull in cultivated rice. Transgenic study indicated that Bh4 could restore the black pigment on hulls in cv Guangluai 4 and Kasalath. Bh4 sequence alignment of all taxa with the outgroup Oryza barthii showed that the wild rice maintained comparable levels of nucleotide diversity that were about 70 times higher than those in the cultivated rice. The results from the maximum likelihood Hudson-Kreitman-Aguade test suggested that the significant reduction in nucleotide diversity in rice cultivars could be caused by artificial selection. We propose that the straw-white hull was selected as an important visual phenotype of nonshattered grains during rice domestication.     

G-quadruplex DNA aptamers generated for systemin

Ligands specific to bioactive molecules play important roles in biomedical researches and applications, such as biological assay, diagnosis and therapy. Systemin is a peptide hormone firstly identified in plant. In this paper we report the selection of a group of DNA aptamers that can specifically bind to systemin. Through comparing the predicted secondary structures of all the aptamers, a hairpin structure with G-rich loop was determined to be the binding motif of these aptamers. The G-rich loop region of this binding motif was further characterized to fold into an antiparallel G-quadruplex by truncation-mutation assay and CD spectrum. The apparent equilibrium dissociation constant (K(d)) of one strong binding sequence (S-5-1) was measured to be 0.5 μM. The specificity assay shows that S-5-1 strongly bind to whole systemin, weakly bind to truncated or mutated systemin and does not bind to the scrambled peptide with the same amino acid composition as systemin. The high affinity and specificity make S-5-1 hold potentials to serve as a molecular ligand applied in detection, separation and functional investigation of systemin in plants.     

Expression and secretion of a single-chain sweet protein monellin in <Emphasis Type="Italic">Bacillus subtilis</Emphasis> by <Emphasis Type="Italic">sacB</Emphasis> promoter and signal peptide

The sweet protein monellin gene was expressed in Bacillus subtilis under the control of the Bacillus subtilis sacB promoter and signal peptide sequence. A 294-bp DNA fragment, coding for sweet protein monellin, was ligated into the Escherichia coli/B. subtilis shuttle vector pHPC, producing pHPMS, which was subsequently transformed into B. subtilis QB1098, DB104, and DB403. The peptide efficiently directed the secretion of monellin from the recombinant B. subtilis cells. A maximum yield of monellin of 0.29 g protein l⁻¹ was obtained from the supernatant of B. subtilis DB403 harboring pHPMS. SDS-PAGE confirmed the purity of the recombinant product.     

Analysis of expressed sequence tags from grapevine flower and fruit and development of simple sequence repeat markers

A total of 6,230 EST sequences were produced from 7,561 clones in a cDNA library generated from grapevine (Vitis vinifera cv. ‘Summer Black’) flower and fruit tissues in this study. After cluster and assembly analysis of the datasets, 3,582 unigenes (GenBank accession numbers GW836604–GW840185) were established, among which 381 were new grapevine EST sequences. Out of the 381 new ESTs, 289 could be mapped on the 19 grapevine chromosomes. 913 unique ESTs with known or putative functions were assigned to 11 putative cellular roles. 540 potentially workable grapevine EST-SSRs were developed from 3,582 unigenes and about 42.6% of these unigenes were identified as true-to-type SSR loci and could amplify polymorphic bands from 22 individual plants of V. vinifera L, indicating that grapevine EST datasets are a valuable source for the development of functional simple sequence repeat (SSR) markers.     

Specific leaf area, leaf nitrogen content, and photosynthetic acclimation of Trifolium repens L. seedlings grown at different irradiances and nitrogen concentrations

Clover seedlings were grown at different nitrogen concentrations (5, 10, 15, 20, 25 mM NO₃ ⁻, i.e. N₅ to N₂₅) and two irradiances, I (200 and 400 μmol m⁻² s⁻¹ of photon flux density, i.e. I ₂₀₀ and I ₄₀₀). Net photosynthetic rate (P _N), photosynthetic nitrogen use efficiency (PNUE), leaf chlorophyll (Chl) content, maximum photochemical efficiency (F_v/F_m), and actual photochemical efficiency of photosystem 2 (PS2) (Φ_PS2) increased from N₅ to N₁₅ and decreased with N₁₅ to N₂₅. P _N, PNUE, and Φ_PS2 were higher at I ₄₀₀ than at I ₂₀₀, but F_v/F_m and leaf Chl contents at I ₄₀₀ were lower than at I ₂₀₀. The effects of the N and I on specific leaf area (SLA) and N contents per unit dry mass (N_m) were similar, the SLA and N_m increased from N₅ to N₂₅ and they were higher at I ₂₀₀ than at I ₄₀₀. The nitrogen contents per unit area (N_a) increased from N₅ to N₂₀, but decreased from N₂₀ to N₂₅. The N_a was higher at I ₂₀₀ than at I ₄₀₀ when Trifolium repens grew at N₅ and N₁₀, but it was higher at I ₄₀₀ than at I ₂₀₀ at N₁₅ to N₂₅.     

Abscisic acid,sucrose, and auxin coordinately regulate berry ripening process of the Fujiminori grape

The aim of this study was to examine the effect of abscisic acid (ABA), sucrose, and auxin on grape fruit development and to assess the mechanism of these three factors on the grape fruit ripening process. Different concentrations of ABA, sucrose, and auxin were used to treat the grape fruit, and the ripening-related indices, such as physiological and molecular level parameters, were analyzed. The activity of BG protein activity was analyzed during the fruit development. Sucrose, ABA, and auxin influenced the grape fruit sugar accumulation in different ways, as well as the volatile compounds, anthocyanin content, and fruit firmness. ABA and sucrose induced, but auxin blocked, the ripening-related gene expression levels, such as softening genes PE, PG, PL, and CELL, anthocyanin genes DFR, CHI, F3H, GST, CHS, and UFGT, and aroma genes Ecar, QR, and EGS. ABA, sucrose, and glucose induced the fruit dry weight accumulation, and auxin mainly enhanced fruit dry weight through seed weight accumulation. In the early development of grape, starch was the main energy storage; in the later, it was glucose and fructose. Sucrose metabolism pathway-related gene expression levels were significant for glucose and fructose accumulation. BG protein activity was important in the regulation of grape ABA content levels. ABA plays a core role in the grape fruit development; sucrose functions in fruit development through two pathways: one was ABA dependent, the other ABA independent. Auxin blocked ABA accumulation to regulate the fruit development process.