A maize semidwarf mutant reveals a GRAS transcription factor involved in brassinosteroid signaling.

Brassinosteroids (BR) and gibberellins (GA) regulate plant height and leaf angle in maize (Zea mays). Mutants with defects in BR or GA biosynthesis or signaling identify components of these pathways and enhance our knowledge about plant growth and development. In this study, we characterized three recessive mutant alleles of GRAS transcription factor 42 (gras42) in maize, a GRAS transcription factor gene orthologous to the DWARF AND LOW TILLERING (DLT) gene of rice (Oryza sativa). These maize mutants exhibited semi-dwarf stature, shorter and wider leaves, and more upright leaf angle. Transcriptome analysis revealed a role for GRAS42 as a determinant of BR signaling. Analysis of the expression consequences from loss of GRAS42 in the gras42-mu1021149 mutant indicated a weak loss of BR signaling in the mutant, consistent with its previously demonstrated role in BR signaling in rice. Loss of BR signaling was also evident by the enhancement of weak BR biosynthetic mutant alleles in double mutants of nana plant1-1 and gras42-mu1021149. The gras42-mu1021149 mutant had little effect on GA-regulated gene expression, suggesting that GRAS42 is not a regulator of core GA signaling genes in maize. Single cell expression data identified gras42 expressed among cells in the G2/M phase of the cell cycle consistent with its previously demonstrated role in cell cycle gene expression in Arabidopsis (Arabidopsis thaliana). Cis-acting natural variation controlling GRAS42 transcript accumulation was identified by expression genome-wide association study (eGWAS) in maize. Our results demonstrate a conserved role for GRAS42/SCARECROW-LIKE 28 (SCL28)/DLT in BR signaling, clarify the role of this gene in GA signaling, and suggest mechanisms of tillering and leaf angle control by BR.

Molecular Insights into the Physical Adsorption of Amphiphilic Protein PhaF onto Copolyester Surfaces.

Phasins are amphiphilic proteins located at the polymer-cytoplasm interface of bacterial polyhydroxyalkanoates (PHA). The immobilization of phasins on biomaterial surfaces is a promising way to enhance the hydrophilicity and supply cell-directing elements in bioinstructing processes. Optimizing the physical adsorption of phasins requires deep insights into molecular processes during polymer-protein interactions to preserve their structural conformation while optimizing surface coverage. Here, the assembly, organization, and stability of phasin PhaF from Pseudomonas putida at interfaces is disclosed. The Langmuir technique, combined with in situ microscopy and spectroscopic methods, revealed that PhaF forms stable and robust monolayers at different temperatures, with an almost flat orientation of its α-helix at the air-water interface. PhaF adsorption onto preformed monolayers of poly[(3-R-hydroxyoctanoate)-co-(3-R-hydroxyhexanoate)] (PHOHHx), yields stable mixed layers below π = ∼15.7 mN/m. Further insertion induces a molecular reorganization. PHOHHx with strong surface hydrophobicity is a more adequate substrate for PhaF adsorption than the less hydrophobic poly[(rac-lactide)-co-glycolide] (PLGA). The observed orientation of the main axis of the protein in relation to copolyester interfaces ensures the best exposure of the hydrophobic residues, providing a suitable coating strategy for polymer functionalization.

Langmuir Monolayers as Tools to Study Biodegradable Polymer Implant Materials.

Langmuir monolayers provide a fast and elegant route to analyze the degradation behavior of biodegradable polymer materials. In contrast to bulk materials, diffusive transport of reactants and reaction products in the (partially degraded) material can be neglected at the air-water interface, allowing for the study of molecular degradation kinetics in experiments taking less than a day and in some cases just a few minutes, in contrast to experiments with bulk materials that can take years. Several aspects of the biodegradation behavior of polymer materials, such as the interaction with biomolecules and degradation products, are directly observable. Expanding the technique with surface-sensitive instrumental techniques enables evaluating the evolution of the morphology, chemical composition, and the mechanical properties of the degrading material in situ. The potential of the Langmuir monolayer degradation technique as a predictive tool for implant degradation when combined with computational methods is outlined, and related open questions and strategies to overcome these challenges are pointed out.

Multiplex single-cell quantification of rare RNA transcripts from protoplasts in a model plant system.

Here we demonstrate multiplex and simultaneous detection of four different rare RNA species from plant, Arabidopsis thaliana, using surface-enhanced Raman spectroscopy (SERS) and gold nanoprobes at single-cell resolution. We show the applicability of nanoparticle-based Raman spectroscopic sensor to study intracellular RNA copies. First, we demonstrate that gold-nanoparticles decorated with Raman probes and carrying specific nucleic acid probe sequences can be uptaken by the protoplasts. We confirm the internalization of gold nanoprobes by transmission electron microscopy, inductively-coupled plasma-mass spectrometry and fluorescence imaging. Second, we show the utility of a SERS platform to monitor individual alternatively spliced (AS) variants and miRNA copies within single cells. Finally, the distinctive spectral features of Raman-active dyes were exploited for multiplex analysis of AtPTB2, AtDCL2, miR156a and miR172a. Furthermore, single-cell studies were validated by in vitro quantification and evaluation of nanotoxicity of gold probes. Raman tag functionalized gold nanosensors yielded an approach for the tracking of rare RNAs within the protoplasts. The SERS-based approach for quantification of RNAs has the capability to be a highly sensitive, accurate and discerning method for single-cell studies including AS variants quantification and rare miRNA detection in specific plant species.

nana plant2 Encodes a Maize Ortholog of the Arabidopsis Brassinosteroid Biosynthesis Gene DWARF1, Identifying Developmental Interactions between Brassinosteroids and Gibberellins.

A small number of phytohormones dictate the pattern of plant form affecting fitness via reproductive architecture and the plant's ability to forage for light, water, and nutrients. Individual phytohormone contributions to plant architecture have been studied extensively, often following a single component of plant architecture, such as plant height or branching. Both brassinosteroid (BR) and gibberellin (GA) affect plant height, branching, and sexual organ development in maize (Zea mays). We identified the molecular basis of the nana plant2 (na2) phenotype as a loss-of-function mutation in one of the two maize paralogs of the Arabidopsis (Arabidopsis thaliana) BR biosynthetic gene DWARF1 (DWF1). These mutants accumulate the DWF1 substrate 24-methylenecholesterol and exhibit decreased levels of downstream BR metabolites. We utilized this mutant and known GA biosynthetic mutants to investigate the genetic interactions between BR and GA. Double mutants exhibited additivity for some phenotypes and epistasis for others with no unifying pattern, indicating that BR and GA interact to affect development but in a context-dependent manner. Similar results were observed in double mutant analyses using additional BR and GA biosynthetic mutant loci. Thus, the BR and GA interactions were neither locus nor allele specific. Exogenous application of GA3 to na2 and d5, a GA biosynthetic mutant, also resulted in a diverse pattern of growth responses, including BR-dependent GA responses. These findings demonstrate that BR and GA do not interact via a single inclusive pathway in maize but rather suggest that differential signal transduction and downstream responses are affected dependent upon the developmental context.

Single molecule Raman spectroscopic assay to detect transgene from GM plants.

Substantial concerns have been raised for the safety of transgenics on human health and environment. Many organizations, consumer groups, and environmental agencies advocate for stringent regulations to avoid transgene products' contamination in food cycle or in nature. Here we demonstrate a novel approach using surface enhanced Raman spectroscopy (SERS) to detect and quantify transgene from GM plants. We show a highly sensitive and accurate quantification of transgene DNA from multiple transgenic lines of Arabidopsis. The assay allows us to detect and quantify the transgenes as low as 0.10 pg without need for PCR-amplification. This technology is relatively cheap, quick, simple, and suitable for detection at low target concentration.

Enzymatic Degradation of Oligo(ε-caprolactone)s End-Capped with Phenylboronic Acid Derivatives at the Air-Water Interface.

The influence of terminal functionalization of oligo(ε-caprolactone)s (OCL) with phenylboronic acid pinacol ester or phenylboronic acid on the enzymatic degradation behavior at the air-water interface is investigated by the Langmuir monolayer degradation technique. While the unsubstituted OCL immediately degrades after injection of the enzyme lipase from Pseudomonas cepacia, enzyme molecules are incorporated into the films based on end-capped OCL before degradation. This incorporation of enzymes does not inhibit or suppress the film degradation, but retards it significantly. A specific binding of lipase to the polymer monolayer allows studying the enzymatic activity of bound proteins and the influence on the degradation process. The functionalization of a macromolecule with phenyl boronic acid groups is an approach to investigate their interactions with diol-containing biomolecules like sugars and to monitor their specified impact on the enzymatic degradation behavior at the air-water interface.

Induced variations in brassinosteroid genes define barley height and sturdiness, and expand the green revolution genetic toolkit.

Reduced plant height and culm robustness are quantitative characteristics important for assuring cereal crop yield and quality under adverse weather conditions. A very limited number of short-culm mutant alleles were introduced into commercial crop cultivars during the Green Revolution. We identified phenotypic traits, including sturdy culm, specific for deficiencies in brassinosteroid biosynthesis and signaling in semidwarf mutants of barley (Hordeum vulgare). This set of characteristic traits was explored to perform a phenotypic screen of near-isogenic short-culm mutant lines from the brachytic, breviaristatum, dense spike, erectoides, semibrachytic, semidwarf, and slender dwarf mutant groups. In silico mapping of brassinosteroid-related genes in the barley genome in combination with sequencing of barley mutant lines assigned more than 20 historic mutants to three brassinosteroid-biosynthesis genes (BRASSINOSTEROID-6-OXIDASE, CONSTITUTIVE PHOTOMORPHOGENIC DWARF, and DIMINUTO) and one brassinosteroid-signaling gene (BRASSINOSTEROID-INSENSITIVE1 [HvBRI1]). Analyses of F2 and M2 populations, allelic crosses, and modeling of nonsynonymous amino acid exchanges in protein crystal structures gave a further understanding of the control of barley plant architecture and sturdiness by brassinosteroid-related genes. Alternatives to the widely used but highly temperature-sensitive uzu1.a allele of HvBRI1 represent potential genetic building blocks for breeding strategies with sturdy and climate-tolerant barley cultivars.

Influence of Diurethane Linkers on the Langmuir Layer Behavior of Oligo[(rac-lactide)-co-glycolide]-based Polyesterurethanes.

Three oligo[(rac-lactide)-co-glycolide] based polyesterurethanes (OLGA-PUs) containing different diurethane linkers are investigated by the Langmuir monolayer technique and compared to poly[(rac-lactide)-co-glycolide] (PLGA) to elucidate the influence of the diurethane junction units on hydrophilicity and packing motifs of these polymers at the air-water interface. The presence of diurethane linkers does not manifest itself in the Langmuir layer behavior both in compression and expansion experiments when monomolecular films of OLGA-PUs are spread on the water surface. However, the linker retard the evolution of morphological structures at intermediate compression level under isobaric conditions (with a surface pressure greater than 11 mN m-1 ) compared to the PLGA, independent on the chemical structure of the diurethane moiety. The layer thicknesses of both OLGA-PU and PLGA films decrease in the high compression state with decreasing surface pressure, as deduced from ellipsometric data. All films must be described with the effective medium approximation as water swollen layers.

Effect of T-DNA insertions on mRNA transcript copy numbers upstream and downstream of the insertion site in Arabidopsis thaliana explored by surface enhanced Raman spectroscopy.

We report the effect of a T-DNA insertion on the expression level of mRNA transcripts of the TWISTED DWARF 1 (TWD1) gene upstream and downstream of the T-DNA insertion site in Arabidopsis. A novel approach based on surface-enhanced Raman spectroscopy (SERS) was developed to detect and quantify the effect of a T-DNA insertion on mRNA transcript accumulation at 5'- and 3'-ends of the TWD1 gene. A T-DNA insertion mutant in the TWD1 gene (twd1-2) was chosen to test the sensitivity and the feasibility of the approach. The null mutant of the FK506-like immunophilin protein TWD1 in Arabidopsis shows severe dwarfism and strong disoriented growth of plant organs. A spontaneous arising suppressor allele of twd1-2 called twd-sup displayed an intermediate phenotype between wild type and the knockout phenotype of twd1-2. Both twd1 mutant alleles have identical DNA sequences at the TWD1 locus including the T-DNA insertion in the fourth intron of the TWD1 gene but they show clear variability in the mutant phenotype. We present here the development and application of SERS-based mRNA detection and quantification using the expression of the TWD1 gene in wild type and both mutant alleles. The hallmarks of our SERS approach are a robust and fast assay to detect up to 0.10 fm of target molecules including the ability to omit in vitro transcription and amplification steps after RNA isolation. Instead we perform direct quantification of RNA molecules. This enables us to detect and quantify rare RNA molecules at high levels of precision and sensitivity.

Brassinosteroid control of sex determination in maize.

Brassinosteroids (BRs) are plant hormones that regulate growth and development. They share structural similarities with animal steroids, which are decisive factors of sex determination. BRs are known to regulate morphogenesis and environmental stress responses, but their involvement in sex determination in plants has been only speculative. We show that BRs control sex determination in maize revealed through characterization of the classical dwarf mutant nana plant1 (na1), which also feminizes male flowers. na1 plants carry a loss-of-function mutation in a DET2 homolog--a gene in the BR biosynthetic pathway. The mutant accumulates the DET2-specific substrate (24R)-24-methylcholest-4-en-3-one with a concomitant decrease of downstream BR metabolites. Treatment of wild-type maize plants with BR biosynthesis inhibitors completely mimicked both dwarf and tasselseed phenotypes of na1 mutants. Tissue-specific na1 expression in anthers throughout their development supports the hypothesis that BRs promote masculinity of the male inflorescence. These findings suggest that, in the monoecious plant maize, BRs have been coopted to perform a sex determination function not found in plants with bisexual flowers.

Impedance and morphology of hydroxy- and chloro-functionalized poly(3,4-propylenedioxythiophene) nanostructures.

The new 3,4-propylenedioxythiophenes (ProDOT) bearing hydroxy- or chloro-functionalized side chains of varying length and polarity were synthesized and electropolymerized on single carbon fiber microelectrode (SCFME) using cyclo-voltammetry. Electrochemical impedance spectroscopy (EIS) revealed highest capacitance values for the hydroxy-functionalized Poly 5 carrying a side chain of medium length. The EIS data were fitted with an equivalent electrical circuit giving a good correlation. AFM analysis of the topography showed higher roughness values for Poly 5 than for the two other polymers bearing longer side chains. Due to their reactive end groups the polymers should be useful for post-polymerization functionalization of the electrode surface.

Mutation of the nuclear pore complex component, aladin1, disrupts asymmetric cell division in Zea mays (maize).

The nuclear pore complex (NPC) regulates the movement of macromolecules between the nucleus and cytoplasm. Dysfunction of many components of the NPC results in human genetic diseases, including triple A syndrome (AAAS) as a result of mutations in ALADIN. Here, we report a nonsense mutation in the maize ortholog, aladin1 (ali1-1), at the orthologous amino acid residue of an AAAS allele from humans, alters plant stature, tassel architecture, and asymmetric divisions of subsidiary mother cells (SMCs). Crosses with the stronger nonsense allele ali1-2 identified complex allele interactions for plant height and aberrant SMC division. RNA-seq analysis of the ali1-1 mutant identified compensatory transcript accumulation for other NPC components as well as gene expression consequences consistent with conservation of ALADIN1 functions between humans and maize. These findings demonstrate that ALADIN1 is necessary for normal plant development, shoot architecture, and asymmetric cell division in maize.

Arabidopsis brassinosteroid biosynthetic mutant dwarf7-1 exhibits slower rates of cell division and shoot induction.

BACKGROUND: Plant growth depends on both cell division and cell expansion. Plant hormones, including brassinosteroids (BRs), are central to the control of these two cellular processes. Despite clear evidence that BRs regulate cell elongation, their roles in cell division have remained elusive. RESULTS: Here, we report results emphasizing the importance of BRs in cell division. An Arabidopsis BR biosynthetic mutant, dwarf7-1, displayed various characteristics attributable to slower cell division rates. We found that the DWARF4 gene which encodes for an enzyme catalyzing a rate-determining step in the BR biosynthetic pathways, is highly expressed in the actively dividing callus, suggesting that BR biosynthesis is necessary for dividing cells. Furthermore, dwf7-1 showed noticeably slower rates of callus growth and shoot induction relative to wild-type control. Flow cytometric analyses of the nuclei derived from either calli or intact roots revealed that the cell division index, which was represented as the ratio of cells at the G2/M vs. G1 phases, was smaller in dwf7-1 plants. Finally, we found that the expression levels of the genes involved in cell division and shoot induction, such as PROLIFERATING CELL NUCLEAR ANTIGEN2 (PCNA2) and ENHANCER OF SHOOT REGENERATION2 (ESR2), were also lower in dwf7-1 as compared with wild type. CONCLUSIONS: Taken together, results of callus induction, shoot regeneration, flow cytometry, and semi-quantitative RT-PCR analysis suggest that BRs play important roles in both cell division and cell differentiation in Arabidopsis.

Insights into the formation and operation of polyaniline sulfonate/cytochrome c multilayer electrodes: contributions of polyelectrolytes' properties.

The multilayer formation of two different sulfonated polyanilines with cytochrome c is presented and mechanistic aspects of the contributions of the polyelectrolytes' properties to the characteristics of the assemblies are discussed. These two modified polymers, PASA1 and PASA2 are chemically synthesized and differ in the grade of sulfonation, substitution, and the chain length of the polymer. The influence of these properties on the multilayer assembly with cytochrome c is studied in detail by Quartz Crystal Microbalance (QCM) technique and Cyclic Voltammetry (CV). It is shown that the multilayer formation is successful, however, the redox activity of polyanilines itself has to be taken into account. In the case of a strong redox activity (PASA2) voltammetric analysis allows the separation of redox processes addressed to the polyelectrolyte and cyt c. For multilayers with PASA1 as building block electroactivity can be predominantly attributed to cyt c ensuring a high amount of electroactive protein and a low probability for interfering redox reaction, making this system suitable for biosensor applications.

Nanofiber network of electropolymerized 3,4-(2-benzylpropylenedioxy)thiophene on single carbon fiber microelectrode.

Electropolymerization of 3,4-(2-benzylpropylene)-dioxythiophene (ProDOT-Bz) on (approximately 7 microm diameter) single carbon fiber microelectrodes (SCFMEs) in different electrolytes resulted the network of nanofiber structure. Electropolymerization performed in different electrolytes by using cyclovoltammetric technique. Surface morphology of coatings was investigated by Scanning Electron Microscopy (SEM), the attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) was used for the characterization. A linear relationship between peak currents of films with polymerization charge, and capacitances and coating thickness were obtained. The size of nanofiber network (and pores) can be controlled by the scan rate of the cyclovoltammetric process. Electrochemical impedance spectroscopic investigation of these nanostructures has been indicated the capacitive behavior of electrode system. Electrochemical Impedance Spectroscopic characterization (Capacitance) of the PProDOTBz/SCFME consisting cell was simulated at applied potential and parameters explained by equivalent circuit modeling.

Plant ABC proteins--a unified nomenclature and updated inventory.

The ABC superfamily comprises both membrane-bound transporters and soluble proteins involved in a broad range of processes, many of which are of considerable agricultural, biotechnological and medical potential. Completion of the Arabidopsis and rice genome sequences has revealed a particularly large and diverse complement of plant ABC proteins in comparison with other organisms. Forward and reverse genetics, together with heterologous expression, have uncovered many novel roles for plant ABC proteins, but this progress has been accompanied by a confusing proliferation of names for plant ABC genes and their products. A consolidated nomenclature will provide much-needed clarity and a framework for future research.

Effect of electrolyte on the electropolymerization of 2,2-dibutyl-3,4-propylenedioxythiophene on carbon fiber microelectrodes.

Electrocoating of 2,2 dibutylpropylene dioxythiophene on carbon fiber microelectrodes (CFMEs) in different electrolytes in acetonitrile was performed, and surface morphology and electrochemical impedance spectroscopic investigation has been carried out. Impedance spectra showed the typical form of Z(IM) versus Z(RE) for transmission-line at frequencies 10 Hz, with transition to almost pure capacitive behaviour down to 10 mHz (the lower limit of frequency scan).