Proteomic analysis of differential anther development from sterile/fertile lines in Capsicum annuum L.

Background: Pepper (Capsicum annuum L.) is a major cash crop throughout the world. Male sterility is an important characteristic in crop species that leads to a failure to produce functional pollen, and it has crucial roles in agricultural breeding and the utilization of heterosis. Objectives: In this study, we identified many crucial factors and important components in metabolic pathways in anther and pollen development, and elucidated the molecular mechanism related to pollen abortion in pepper. Methods: Pepper pollen was observed at different stages to detect the characteristics associated with male sterility and fertility. The phytohormone and oxidoreductase activities were detected in spectrophotometric and redox reaction assays, respectively. Proteins were extracted from male sterile and fertile pepper lines, and identified by TMT/iTRAQ (tandem mass tags/isobaric tags for relative and absolute quantitation) and LC-MS/MS (liquid chromatograph-mass spectrometer) analysis. Differentially abundant proteins (DAPs) were analyzed based on Gene Ontology annotations and the Kyoto Encyclopedia of Genes and Genomes database according to |fold change)| > 1.3 and P value < 0.05. DAPs were quantified in the meiosis, tetrad, and binucleate stages by parallel reaction monitoring (PRM). Results: In this study, we screened and identified one male sterile pepper line with abnormal cytological characteristics in terms of pollen development. The peroxidase and catalase enzyme activities were significantly reduced and increased, respectively, in the male sterile line compared with the male fertile line. Phytohormone analysis demonstrated that the gibberellin, jasmonic acid, and auxin contents changed by different extents in the male sterile pepper line. Proteome analysis screened 1,645 DAPs in six clusters, which were mainly associated with the chloroplast and cytoplasm based on their similar expression levels. According to proteome analysis, 45 DAPs were quantitatively identified in the meiosis, tetrad, and binucleate stages by PRM, which were related to monoterpenoid biosynthesis, and starch and sucrose metabolism pathways. Conclusions: We screened 1,645 DAPs by proteomic analysis and 45 DAPs were related to anther and pollen development in a male sterile pepper line. In addition, the activities of peroxidase and catalase as well as the abundances of phytohormones such as gibberellin, jasmonic acid, and auxin were related to male sterility. The results obtained in this study provide insights into the molecular mechanism responsible for male sterility and fertility in pepper.

Influence of Allyl Isothiocyanate on the Soil Microbial Community Structure and Composition during Pepper Cultivation.

Allyl isothiocyanate (AITC), as a fumigant, plays an important role in soil control of nematodes, soilborne pathogens, and weeds, but its effects on soil microorganisms are unclear. In this study, the effects of AITC on microbial diversity and community composition of Capsicum annuum L. soil were investigated through Illumina high-throughput sequencing. The results showed that microbial diversity and community structure were significantly influenced by AITC. AITC reduced the diversity of soil bacteria, stimulated the diversity of the soil fungal community, and significantly changed the structure of fungal community. AITC decreased the relative abundance of dominant bacteria Planctomycetes, Acinetobacter, Pseudodeganella, and RB41, but increased that of Lysobacter, Sphingomonas, Pseudomonas, Luteimonas, Pseudoxanthomonas, and Bacillus at the genera level, while for fungi, Trichoderma, Neurospora, and Lasiodiplodia decreased significantly and Aspergillus, Cladosporium, Fusarium, Penicillium, and Saccharomyces were higher than the control. The correlation analysis suggested cellulase had a significant correlation with fungal operational taxonomic units and there was a significant correlation between cellulase and fungal diversity, while catalase, cellulose, sucrase, and urease were the major contributors in the shift of the community structure. Our results will provide useful information for the use of AITC in the assessment of environmental and ecological security.

Synergistic effects of organic fertilizer and corn straw on microorganisms of pepper continuous cropping soil in China.

Because of the large population, large demand, limited arable land and many environmental factors, continuous cropping have become a very common phenomenon in China. However, long-term continuous cropping has caused a series of serious soil-borne diseases, and the yield and quality of crops to drop, which seriously restricted the sustainable development of agricultural industry. Therefore, in order to improve the yield of pepper and reduce the occurrence of soil-borne diseases, it is essential to understand the effect of continuous cropping of pepper on soil microbial community composition and abundance. In this study, high throughput sequencing was used to study the effects of seven treatments of organic fertilizers and corn straw on soil microbial community and function of pepper continuous cropping. The results showed that the yield of all treatments was significantly higher than that of the control. The soil microbial diversity and community composition showed that Proteobacteria and Ascomycota were the most abundant phylum in all treatments. In conclusion, there were significant differences among the seven treatments and the treatment of fowl dung with corn straw was the best fertilizer combination to improve the yield and output value of pepper. Besides, the addition of fowl dung and corn straw not only can improve the community and functions of microorganisms, but also enhance the ability of disease resistance, and ultimately decrease the soil-borne diseases. The results will help to provide scientific basis for rational application of organic fertilizer and corn straw, and overcoming continuous cropping obstacles.

Vaspin regulates the osteogenic differentiation of MC3T3-E1 through the PI3K-Akt/miR-34c loop.

Vaspin (visceral adipose tissue-derived serine protease inhibitor) is a newly discovered adipokine that widely participates in diabetes mellitus, polycystic ovarian syndrome and other disorders of metabolism. However, the effect of vaspin on the regulation of osteogenesis and the mechanism responsible are still unclear. Here, we found that vaspin can attenuate the osteogenic differentiation of the preosteoblast cell line MC3T3-E1 in a dose-dependent way; also, during this process, the expression of miRNA-34c (miR-34c) was significantly increased. Down-regulation of the expression of miR-34c in MC3T3-E1 diminished the osteogenic inhibitory effect of vaspin, while the up-regulation of miR-34c increased this effect through its target gene Runx2. Meanwhile, we found that vaspin could also activate the PI3K-Akt signalling pathway. Blocking the PI3K-Akt signalling pathway with specific inhibitors could decrease the osteogenic inhibitory effect of vaspin as well as the expression level of miR-34c. Furthermore, knock-down of miR-34c could promote the activation of Akt, which was probably realised by targeting c-met expression. Thus, PI3K-Akt and miR-34c constituted a modulation loop and controlled the expression of each other. Taken together, our study showed that vaspin could inhibit the osteogenic differentiation in vitro, and the PI3K-Akt/miR-34c loop might be the underlying mechanism.

Ginkgolide B Reduces the Degradation of Membrane Phospholipids to Prevent Ischemia/Reperfusion Myocardial Injury in Rats.

Platelet-activating factor (PAF), a bioactive phospholipid, plays an important role in the integrity of the cellular membrane structure, and is involved in the pathogenesis of myocardial ischemia/reperfusion (IR) injuries. In this study, we tested the hypothesis that blockage of PAF receptor by BN 52021 (Ginkgolide B) can prevent IR-induced degradation of the myocardial membrane phospholipid, and deterioration of the cardiac function. Rat hearts in situ were subjected to 5 min ischemia and followed by 10 min reperfusion. Cardiac performances during periods of ischemia and reperfusion were monitored, and the amount of membrane phospholipids was analyzed. Myocardial total phospholipids, phosphatidylcholine, and phosphatidylethanolamine were decreased significantly in ischemia-reperfusion rat hearts compared with those of sham-operated rat hearts. Degradation of the membrane phospholipid was accompanied by the deterioration of cardiac functions and increase in serum lactate dehydrogenase (LDH) activity. BN 52021 (15 mg/kg), given by intravenous infusion 10 min prior to the left anterior descending coronary artery occlusion, reduced IR-related degradation of the myocardial phospholipids, the activity of serum LDH, and was concomitant with improvement of cardiac function. Furthermore, we demonstrated that the production of PAF was increased and BN 52021 decreased cellular damage in cultured anoxic cardiomyocytes. These results indicated that PAF antagonist BN 52021 has a protective effect against IR-induced myocardial dysfunction and degradation of the membrane phospholipids.

Conductivity and electrophoretic mobility of dilute ionic solutions.

Two complementary continuum theories of electrokinetic transport are examined with particular emphasis on the equivalent conductance of binary electrolytes. The "small ion" model [R.M. Fuoss, L. Onsager, J. Phys. Chem. 61 (1957) 668] and "large ion" model [R.W. O'Brien, L.R. White, J. Chem. Soc. Faraday Trans. 2 (74) (1978) 1607] are both discussed and the "large ion" model is generalized to include an ion exclusion distance and to account in a simple but approximate way for the Brownian motion of all ions present. In addition, the "large ion" model is modified to treat "slip" hydrodynamic boundary conditions in addition to the standard "stick" boundary condition. Both models are applied to the equivalent conductance of dilute KCl, MgCl(2), and LaCl(3) solutions and both are able to reproduce experimental conductances to within an accuracy of several tenths of a percent. Despite fundamental differences in the "small ion" and "large ion" theories, they both work equally well in this application. In addition, both "stick-large ion" and "slip-large ion" models are equally capable of accounting for the equivalent conductances of the three electrolyte solutions.

Modeling the electrophoresis and transport of peptides: the effective sphere model and complex formation.

Modeling the electrophoretic mobility of peptides is examined in this study using a "coarse grained" bead model, or B model for short 8 and also a simpler "effective sphere" (ES) model. A comparison between the B and ES models is carried out for peptide models covering a broad range of ionic strength, peptide charge, and peptide length. At ionic strengths lower than approximately 0.013 M, the B and ES models agree to within a few percent. The ES model is much simpler than the B model and is of particular value in certain applications such as complex formation between peptide and other species in the BGE. The mobility behavior of oligoglycine in a borate buffer at high pH can be accounted for when complex formation is included in modeling.

Modeling the electrophoresis of oligoglycines.

The electrophoretic mobility of low molecular mass oligoglycines is examined in this study using a "coarse-grained" bead modeling methodology [Pei, H., Allison, S. A., J. Chromatogr. A 2009, 1216, 1908-1916]. The advantage of focusing on these peptides is that their charge state is well known [Plasson, R., Cottet, H., Anal. Chem. 2006, 78, 5394-5402] and extensive electrophoretic mobility data are also available in different buffers [Survay, M. A., Goodall, D. M., Wren, S. A. C., Rowe, R. C., J. Chromatogr. A 1996, 741, 99-113] and over a broad range of temperatures [Plasson, R., Cottet, H., Anal Chem. 2005, 77, 6047-6054]. Except for assumptions about peptide secondary structure, the B model has no adjustable parameters. It is concluded that the oligoglycines adopt a random configuration at high temperature (50 degrees C and higher), but more compact conformations at lower temperature. It is proposed that triglycine through pentaglycine adopt compact cyclic structures at low temperature (up to about 25 degrees C) in aqueous solution. At 25 degrees C, buffer interactions are also examined and may or may not influence peptide conformation depending on the buffer species. In a borate buffer at high pH, the mobility data are consistent with complex formation between the oligoglycine and borate anion.

The dependence of the electrophoretic mobility of small organic ions on ionic strength and complex formation.

The ionic strength dependence of the electrophoretic mobility of small organic anions with valencies up to -3 is investigated in this study. Provided the anions are not too aspherical, it is argued that shape and charge distribution have little influence on mobility. To a good approximation, the electrophoretic mobility of a small particle should be equal to that of a model sphere with the same hydrodynamic radius and same net charge. For small ions, the relaxation effect (distortion of the ion atmosphere from equilibrium due to external electric and flow fields) is significant even for monovalent ions. Alternative procedures of accounting for the relaxation effect are examined. In order to account for the ionic strength dependence of a specific set of nonaromatic and aromatic anions in aqueous solution, it is necessary to include complex formation between the anion with species in the BGE. A number of possible complexes are considered. When the BGE is Tris-acetate, the most important of these involves the complex formed between anion and Tris, the principle cation in the BGE. When the BGE is sodium borate, an anion-anion (borate) complex appears to be important, at least when the organic anion is monovalent. An algorithm is developed to analyze the ionic strength dependence of the electrophoretic mobility. This algorithm is applied to two sets of organic anions from two independent research groups.

Viscosity of dilute model bead arrays at low shear: inclusion of short range solute-solvent interactions.

The intrinsic viscosity, [eta], of certain polymer-solvent systems, such as alkanes in benzene, are "anomalous" in the sense that [eta] for low molecular weight fractions are low and in certain cases negative (Dewan, K. K.; Bloomfield, V. A.; Berget, P. G.; J. Phys. Chem. 1974, 75, 3120). In this work, the theory of the viscosity of a dilute suspension of macromolecules at low shear is formulated that accounts for possible solute-solvent interactions. In doing so, we show that negative intrinsic viscosities are possible and are able to reproduce quite well the known length dependence of [eta] for alkanes in benzene. The coarse grained, solvent continuum, bead model developed here is an extension of previous work (Allison, S. A.; Pei, H. J. Phys. Chem. B 2009, 113, 8056). Following Fixman (Fixman, M. J. Chem. Phys. 1990, 92, 6858), we assume that solute-solvent interactions are short-range in character and can be separated from long-range hydrodynamic interactions between different beads. These interactions are accounted for by introducing three adjustable parameters specific to the transport of small "monomeric" solutes in the solvent of interest. Long range hydrodynamic interactions are accounted for to order a(J)(2)/r(IJ)(3) (a(J) is a bead radius and r(IJ) is an interbead distance). In modeling a macromolecule as an arbitrary array of N beads, the transport of the array is examined numerically in 5 different elementary shear fields. The most computationally demanding component of the procedure involves the inversion of a 12N by 12N matrix. In the present work, we restrict ourselves to systems with a maximum N of about 100. Our procedure is first applied to short rods and rings of from 2 to 10 beads which can be compared with independent results from the literature. Agreement is found to be better than 5%. Modeling macromolecules as wormlike chains, the procedure is then applied first to duplex DNA and then to alkanes in benzene. In both cases, it is possible to obtain excellent agreement between modeling and experiment.

Translational diffusion constants of short peptides: measurement by NMR and their use in structural studies of peptides.

In this work, pulsed field gradient NMR is used to measure the translational self-diffusion constants (D(T)'s) of five simple peptides (GG, GR, GGR, GGNA, and GGRA) as well as glycine, G, at low concentration. The experiments were carried out in D(2)O at 298 K at pD = 3.5 in 80 mM sodium phosphate buffer. Of the five peptides, four are being reported for the first time (all except GG) and the results of G and GG are compared with D(T)'s from the literature. When corrected for differences in solvent viscosity and temperature, the discrepancy between D(T)'s of G and GG measured in the present work are lower than previously published values by several percent. Given the range of values reported in the literature for specific values of the amino acids by different groups, this discrepancy is regarded as reasonable. Diffusion constants can provide useful information about molecular size and conformation. Modeling a peptide made up of N amino acids as 2N beads (2 for each amino acid present in the peptide), we examine the diffusion constants of the above-mentioned peptides and conclude they are consistent with unfolded or random conformations in solution. Also, by comparing the diffusion constants of G and GG, an estimate of the change in solvation volume due to the loss of a water molecule can be estimated.

Viscosity of dilute suspensions of rigid bead arrays at low shear: accounting for the variation in hydrodynamic stress over the bead surfaces.

In this work, we examine the viscosity of a dilute suspension of irregularly shaped particles at low shear. A particle is modeled as a rigid array of nonoverlapping beads of variable size and geometry. Starting from a boundary element formalism, approximate account is taken of the variation in hydrodynamic stress over the surface of the individual beads. For a touching dimer of two identical beads, the predicted viscosity is lower than the exact value by 5.2%. The methodology is then applied to several other model systems including tetramers of variable conformation and linear strings of touching beads. An analysis is also carried out of the viscosity and translational diffusion of several dilute amino acids and diglycine in water. It is concluded that continuum hydrodynamic modeling with stick boundary conditions is unable to account for the experimental viscosity and diffusion data simultaneously. A model intermediate between "stick" and "slip" could possibly reconcile theory and experiment.

Brownian dynamics simulation of the diffusion of rods and wormlike chains in a gel modeled as a cubic lattice: application to DNA.

The translational diffusion constant of a particle, D, in a congested medium or a gel can be written as the product of two terms that account for long-range hydrodynamic interaction between the gel or congested medium and the particle, DEM, and a short-range "steric" term, S. For particles of arbitrary shape, DEM has been examined previously within the framework of the effective medium, EM, model (S. Allison et al., J. Phys. Chem. B 2008, 112, 5858-5866). In the present work, we examine S for rod- and wormlike chain models of duplex DNA in the size range of 100 to over 2000 base pairs. The gel is modeled explicitly as a cubic lattice, and Brownian dynamics simulation is used to examine S for a wide range of rod/wormlike chain and gel parameters. For wormlike chains with P = 50 nm, an empirical formula is derived for S that should be valid over a wide range of wormlike chain/gel parameters. For duplex DNA in the size of several hundred to several thousand base pairs in an agarose gel of 2% or less, fair agreement between modeling and experiment is obtained. However, modeling overestimates the length dependence of D observed experimentally. Finally, the reduction of D of DNA (100 to over 1000 base pairs in length) in cytoplasm relative to water can be accounted for quite well using the effective medium plus steric correction approach.

The free solution electrophoretic mobility of peptides by a bead modeling methodology.

A bead modeling methodology, BMM, discussed previously to compute the free solution electrophoretic mobility of peptides [H. Pei, Y. Xin, S.A. Allison, J. Sep. Sci. 31 (2008) 554-564], is generalized to avoid the approximation of orientationally preaveraging hydrodynamic interaction. In general, peptide mobilities computed without preaveraging are lower by about 2%. The BMM is then used to study the free solution electrophoretic mobility of several insect oostatic peptides reported previously in a variety of different buffer systems ranging in pH from 2.25 to 8.1 [V. Solinova, V. Kasicka, D. Koval, J. Hlavacek, Electrophoresis, 25 (2004) 2299-2308]. With minor adjustment of the intrinsic pK(a0) of the N-terminal peptide, good agreement between modeling and experiment is achieved for peptide models with random secondary structures in the entire pH range. Model mobilities of these peptides appear to be relatively insensitive to the assumed secondary structure.

Translational diffusion of macromolecules and nanoparticles modeled as non-overlapping bead arrays in an effective medium.

There are three objectives to the present work. First, starting from a boundary element (BE) formulation of low Reynolds number hydrodynamics, model the translational diffusion of macromolecules modeled as an array of non-overlapping beads, and show how this approach is equivalent to previous formulations of "bead hydrodynamics" and under what conditions. Second, show how this approach can be improved upon by accounting for the variation in forces over the surfaces of individual beads and also extending the approach to a gel modeled as an effective medium, EM. Third, develop a "combined obstruction and hydrodynamic effect" model of the translational diffusion of irregularly shaped macromolecules in a gel. In one of the cases studied, the BE approach is shown to be equivalent to previous "bead model" formulations in which intersubunit hydrodynamic interaction is modeled using the Rotne-Prager tensor. A bead model that accounts for the variation in hydrodynamic stress forces over the individual bead surfaces is shown to be in best agreement with exact results for simple bead arrays made up of 2-4 subunits. The translational diffusion of rods, modeled as strings of from 2 to 100 touching beads in dilute gels is examined. Interpolation formulas valid over a range of gel concentrations and rod lengths are derived for the parallel and perpendicular components of the diffusion tensor as well as the orientationally averaged diffusion tensor. The EM model accounts for the long-range hydrodynamic interaction exerted by the gel support matrix on the diffusing particle of interest but does not account for the reduction in diffusion caused by the direct obstruction of the gel, or steric effect. Both effects are accounted for by writing the translational diffusion in a gel as the product of two terms representing long-range hydrodynamic interaction and steric effects. Finally, the diffusion of a 564 base pair DNA in a 2% agarose gel is examined and model results are compared to experiment (Pluen, A.; Netti, P. A.; Jain, R. K.; Berk, D. A. Biophys. J. 1999, 77, 542-552). For reasonable choices of model parameters, fair agreement between theory and experiment is achieved.

Using electrophoretic mobility and bead modeling to characterize the charge and secondary structure of peptides.

Using a modeling methodology developed in our laboratory previously, the free solution electrophoretic mobilities of several peptides are examined to see what they can tell us about: (i) the pK(a)s of specific side groups, and (ii) possible secondary structure. Modeling is first applied to mobility versus pH data of several small peptides (Messana, I. et al., J. Chromatogr. B 1997, 699, 149) where the only adjustable parameter associated with the charge state of the peptide is the pK(a )of the C-terminal. In addition to examining this parameter, the question of possible secondary structure is addressed. For two of the peptides considered, GGNA and GGQA, it is possible to account for the observed mobilities using "random" models with little restriction on the allowed range of Phi-Psi angles. For GGRA and RPPGF, "compact" models (possibly involving an I-turn) must be used to match modeling mobilities with experiment. Finally, three more complicated peptides ranging in size from 15 to 20 amino acids are also examined and characterized (Sitaram, B. R. et al., J. Chromatogr. A 1999, 857, 263). Here also, we find evidence of I-turns or some other "compact" structure in two of the three peptides examined.

Modeling the free solution and gel electrophoresis of biopolymers: the bead array-effective medium model.

Free solution and gel electrophoresis is an extremely useful tool in the separation of biopolymers. The complex nature of biopolymers, coupled with the usefulness of electrophoretic methods, has stimulated the development of theoretical modeling over the last 30 years. In this work, these developments are first reviewed with emphasis on Boundary Element and bead methodologies that enable the investigator to design realistic models of biopolymers. In the present work, the bead methodology is generalized to include the presence of a gel through the Effective Medium model. The biopolymer is represented as a bead array. A peptide, for example, made up of N amino acids is modeled as 2N beads. Duplex DNA is modeled as a discrete wormlike chain consisting of touching beads. The technical details of the method are placed in three Appendices. To illustrate the accuracy and effectiveness of the approach, two applications are considered. Model studies on both the free solution mobility of 73 peptides ranging in size from 2 to 42 amino acids, and the mobility of short duplex DNA in dilute agarose gels are discussed.

Electrophoresis of spheres with uniform zeta potential in a gel modeled as an effective medium.

The effective medium model [H.C. Brinkman, Appl. Sci. Res. A 1 (1947) 27] is used to calculate the electrophoretic mobility of spheres in a gel with uniform zeta potential on their surface. In the absence of a gel support medium or ion relaxation (the distortion of the ion atmosphere from equilibrium due to the presence of an external flow or electric field), our results reduce to those of Henry [D.C. Henry, Proc. R. Soc. London Ser. A 133 (1931) 106]. The relaxation effect can be ignored for weakly charged particles, or for particles with low absolute zeta potential. Using a procedure similar to that employed by O'Brien and White [R.W. O'Brien, L.R. White, J. Chem. Soc. Faraday Trans. 2 74 (1978) 1607], the relaxation effect is accounted for in the present work and results are presented over a wide range of particle sizes, gel concentrations, and zeta potentials in KCl salt solutions. In the limit of no gel, our results reduce to those of earlier investigations. The procedure is then applied to the mobility of Au nanoparticles in agarose gels and model results are compared to recent experiments [D. Zanchet, C.M. Micheel, W.J. Parak, D. Gerion, S.C. Williams, A.P. Alivisatos, J. Phys. Chem. B 106 (2002) 11758; T. Pons, H.T. Uyeda, I.L. Medintz, H. Mattoussi, J. Phys. Chem. B 110 (2006) 20308]. Good agreement with experiment is found for reasonable choices of the model input parameters.