The over-expression of Chrysanthemum crassum CcSOS1 improves the salinity tolerance of chrysanthemum

Soil salinity represents a major constraint on plant growth. Here, we report that the over-expression of the Chrysanthemum crassum plasma membrane Na⁺/H⁺ antiporter gene CcSOS1, driven by the CaMV 35S promoter, improved the salinity tolerance of chrysanthemum ‘Jinba’. In salinity-stressed transgenic plants, both the proportion of the leaf area suffering damage and the electrical conductivity of the leaf were lower in the transgenic lines than in salinity-stressed wild type plants. After a 6 day exposure to 200 mM NaCl, the leaf content of both chlorophyll (a+b) and proline was higher in the transgenic than in the wild type plants. The activity of both superoxide dismutase and peroxidase was higher in the transgenic than in the wild type plants throughout the period of NaCl stress. The transgenic plants had a stronger control over the ingress of Na⁺ into the plant, particularly with respect to the youngest leaves, and so maintained a more favorable K⁺/Na⁺ ratio. The result suggests that a possible strategy for improving the salinity tolerance of chrysanthemum could target the restriction of Na⁺ accumulation. This study is the first to report the transgenic expression of a Na⁺ efflux carrier in chrysanthemum.     

Population shift of binding pocket size and dynamic correlation analysis shed new light on the anticooperative mechanism of PII protein

P_II protein is one of the largest families of signal transduction proteins in archaea, bacteria, and plants, controlling key processes of nitrogen assimilation. An intriguing characteristic for many P_II proteins is that the three ligand binding sites exhibit anticooperative allosteric regulation. In this work, P_II protein from Synechococcus elongatus, a model for cyanobacteria and plant P_II proteins, is utilized to reveal the anticooperative mechanism upon binding of 2‐oxoglutarate (2‐OG). To this end, a method is proposed to define the binding pocket size by identifying residues that contribute greatly to the binding of 2‐OG. It is found that the anticooperativity is realized through population shift of the binding pocket size in an asymmetric manner. Furthermore, a new algorithm based on the dynamic correlation analysis is developed and utilized to discover residues that mediate the anticooperative process with high probability. It is surprising to find that the T‐loop, which is believed to be responsible for mediating the binding of P_II with its target proteins, also takes part in the intersubunit signal transduction process. Experimental results of P_II variants further confirmed the influence of T‐loop on the anticooperative regulation, especially on binding of the third 2‐OG. These discoveries extend our understanding of the P_II T‐loop from being essential in versatile binding of target protein to signal‐mediating in the anticooperative allosteric regulation. Proteins 2014; 82:1048–1059. © 2013 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.     

Role of histidine for charge regulation of unstructured peptides at interfaces and in bulk

Histidine‐rich, unstructured peptides adsorb to charged interfaces such as mineral surfaces and microbial cell membranes. At a molecular level, we investigate the adsorption mechanism as a function of pH, salt, and multivalent ions showing that (1) proton charge fluctuations are—in contrast to the majority of proteins—optimal at neutral pH, promoting electrostatic interactions with anionic surfaces through charge regulation and (2) specific zinc(II)‐histidine binding competes with protons and ensures an unusually constant charge distribution over a broad pH interval. In turn, this further enhances surface adsorption. Our analysis is based on atomistic molecular dynamics simulations, coarse grained Metropolis Monte Carlo, and classical polymer density functional theory. This multiscale modeling provides a consistent picture in good agreement with experimental data on Histatin 5, an antimicrobial salivary peptide. Biological function is discussed and we suggest that charge regulation is a significant driving force for the remarkably robust activity of histidine‐rich antimicrobial peptides. Proteins 2014; 82:657–667. © 2013 Wiley Periodicals, Inc.     

Synthesis of Novel Chiral Tridentate Schiff‐Base Ligands and Their Applications in Catalytic Asymmetric Henry Reaction

A series of chiral tridentate Schiff‐bases were prepared and used as ligands in the catalytic asymmetric Henry reaction. Under the optimal conditions, a variety of arylaldehydes were smoothly converted into corresponding adducts with high yields (up to 98%) and excellent enantioselectivities (up to 97% ee). Chirality 26: 780–783, 2014. © 2014 Wiley Periodicals, Inc.     

Crystal structure of an HD‐GYP domain cyclic‐di‐GMP phosphodiesterase reveals an enzyme with a novel trinuclear catalytic iron centre

Bis‐(3′,5′) cyclic di‐guanylate (c‐di‐GMP) is a key bacterial second messenger that is implicated in the regulation of many crucial processes that include biofilm formation, motility and virulence. Cellular levels of c‐di‐GMP are controlled through synthesis by GGDEF domain diguanylate cyclases and degradation by two classes of phosphodiesterase with EAL or HD‐GYP domains. Here, we have determined the structure of an enzymatically active HD‐GYP domain protein from Persephonella marina (PmGH) alone, in complex with substrate (c‐di‐GMP) and final reaction product (GMP). The structures reveal a novel trinuclear iron binding site, which is implicated in catalysis and identify residues involved in recognition of c‐di‐GMP. This structure completes the picture of all domains involved in c‐di‐GMP metabolism and reveals that the HD‐GYP family splits into two distinct subgroups containing bi‐ and trinuclear metal centres.     

Canola cultivar performance in weed-infested field plots confirms allelopathy ranking from in vitro testing

Crop competition and allelopathy are two cultural control options for possible inclusion in cropping systems. This research aimed to identify superior allelopathic canola genotypes through a two-year field study. First year screening results of 312 diverse Brassica genotypes showed genotypes differed significantly in their ability to suppress weed infestations. Crop plant height was correlated with the competitive ability of several genotypes, while other genotypes showed good weed-suppressive ability despite being short. Thirty-six of the genotypes grown in the field had been previously assessed for their allelopathic ability to inhibit the growth of annual ryegrass (Lolium rigidum) seedlings using an in vitro technique. The highly allelopathic genotypes: Av-opal, Sardi603, Rivette and Atr-beacon performed well against annual ryegrass in the laboratory and also against other species, including Capsella bursa-pastoris, Sisymbrium orientale and Hordeum leporinum in the field. The weakly allelopathic Barossa and X-06-6-3725 genotypes performed poorly both in the laboratory studies and in the field. The following year, field testing of selected genotypes at two sowing dates further suggested that the most allelopathic genotypes in the laboratory bioassay were generally those that suppressed weed numbers and their biomass in the field. The late sowing time increased the natural weed pressure leading to a decrease in both canola grain yield and quality. Many of the highly allelopathic canola genotypes, which caused low weed populations in the field, had relatively low grain yield. This suggests that the allelopathic trait is independent of local adaptation and yields potential under weed-free conditions. Ideally, cultivars with both high allelopathy and high competitive ability would be most useful to help farmers maximise yield and control weeds. Selection for allelopathy in canola shows potential as a future non-chemical weed control option and requires further investigation.     

OsABCB14 functions in auxin transport and iron homeostasis in rice (Oryza sativa L.)

Members of the ATP Binding Cassette B/Multidrug‐Resistance/P–glyco‐protein (ABCB/MDR/PGP) subfamily were shown to function primarily in Oryza sativa (rice) auxin transport; however, none of the rice ABCB transporters have been functionally characterized. Here, we describe that a knock‐down of OsABCB14 confers decreased auxin concentrations and polar auxin transport rates, conferring insensitivity to 2,4‐dichlorophenoxyacetic acid (2,4–D) and indole‐3‐acetic acid (IAA). OsABCB14 displays enhanced specific auxin influx activity in yeast and protoplasts prepared from rice knock‐down alleles. OsABCB14 is localized at the plasma membrane, pointing to an important directionality under physiological conditions. osabcb14 mutants were surprisingly found to be insensitive to iron deficiency treatment (–Fe). Their Fe concentration is higher and upregulation of Fe deficiency‐responsive genes is lower in osabcb14 mutants than in wild‐type rice (Nipponbare, NIP). Taken together, our results strongly support the role of OsABCB14 as an auxin influx transporter involved in Fe homeostasis. The functional characterization of OsABCB14 provides insights in monocot auxin transport and its relationship to Fe nutrition.     

Agrobacterium-mediated genetic transformation and regeneration of transgenic plants using leaf midribs as explants in ramie [Boehmeria nivea (L.) Gaud]

In this study, leaf midribs, the elite explants, were used for the first time to develop an efficient regeneration and transformation protocol for ramie [Boehmeria nivea (L.) Gaud.] via Agrobacterium-mediated genetic transformation. Sensitivity of leaf midribs regeneration to kanamycin was evaluated, which showed that 40 mg l^?1 was the optimal concentration needed to create the necessary selection pressure. Factors affecting the ramie transformation efficiency were evaluated, including leaf age, Agrobacterium concentration, length of infection time for the Agrobacterium solution, acetosyringone concentration in the co-cultivation medium, and the co-cultivation period. The midrib explants from 40-day-old in vitro shoots, an Agrobacterium concentration at OD₆₀₀ of 0.6, 10-min immersion in the bacteria solution, an acetosyringone concentration of 50 mg l^?1 in the co-cultivation medium and a 3-day co-cultivation period produced the highest efficiencies of regeneration and transformation. In this study, the average transformation rate was 23.25 %. Polymerase chain reactions using GUS and NPTII gene-specific primers, Southern blot and histochemical GUS staining analyses further confirmed that the transgene was integrated into the ramie genome and expressed in the transgenic ramie. The establishment of this system of Agrobacterium-mediated genetic transformation and regeneration of transgenic plants will be used not only to introduce genes of interest into the ramie genome for the purpose of trait improvement, but also as a common means of testing gene function by enhancing or inhibiting the expression of target genes.     

Developmental regulations of Perp in mice molar morphogenesis

Angiogenesis, a complex biologic process, is regulated by a large number of angiogenic factors, including vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2). Whether Bone morphogenetic proteins-2 (BMP-2), the osteoinductive factor, could significantly reinforce the effect of VEGF and FGF-2 on angiogenesis has not been studied in detail. To study the positive effects of multiple growth factors on angiogenesis, HUVECs were treated with BMP-2, VEGF, or FGF-2 singly and in binary and ternary combinations. This study further investigates the optimal timing of the ternary combination of BMP-2, VEGF and FGF-2 for angiogenesis in the chorioallantoic membrane (FGF-2 CAM). Results of single applications of BMP-2, VEGF, or FGF-2 suggested that HUVECs angiogenesis could be promoted in a dose-dependent manner and that the optimal concentration of BMP, VEGF and FGF-2 was 10, 50 and 1 ng/mL, respectively. These results indicated that the angiogenic activity of VEGF and FGF-2 was amplified by combining with BMP-2. The ternary combination of BMP-2, VEGF and FGF-2 exhibited a positive and synergistic effect on HUVECs angiogenesis, with the lower concentrations of each factor (1 ng/mL of BMP-2, 25 ng/mL of VEGF and 0.1 ng/mL of FGF-2) being sufficient to show synergistic promotion. When VEGF and FGF-2 were added in the initial activation stage and BMP-2 was added in the maturation stage, both HUVECs angiogenesis in vitro and CAM angiogenesis in vivo could be enhanced more effectively. These results could provide a basis for the controlled release systems capable of delivering multiple factors sequentially to promote angiogenesis in tissue engineering.