Coupling oxidative signals to protein phosphorylation via methionine oxidation in Arabidopsis

The mechanisms involved in sensing oxidative signalling molecules, such as H2O2, in plant and animal cells are not completely understood. In the present study, we tested the postulate that oxidation of Met (methionine) to MetSO (Met sulfoxide) can couple oxidative signals to changes in protein phosphorylation. We demonstrate that when a Met residue functions as a hydrophobic recognition element within a phosphorylation motif, its oxidation can strongly inhibit peptide phosphorylation in vitro. This is shown to occur with recombinant soybean CDPKs (calcium-dependent protein kinases) and human AMPK (AMP-dependent protein kinase). To determine whether this effect may occur in vivo, we monitored the phosphorylation status of Arabidopsis leaf NR (nitrate reductase) on Ser534 using modification-specific antibodies. NR was a candidate protein for this mechanism because Met538, located at the P+4 position, serves as a hydrophobic recognition element for phosphorylation of Ser534 and its oxidation substantially inhibits phosphorylation of Ser534 in vitro. Two lines of evidence suggest that Met oxidation may inhibit phosphorylation of NR-Ser534 in vivo. First, phosphorylation of NR at the Ser534 site was sensitive to exogenous H2O2 and secondly, phosphorylation in normal darkened leaves was increased by overexpression of the cytosolic MetSO-repair enzyme PMSRA3 (peptide MetSO reductase A3). These results are consistent with the notion that oxidation of surface-exposed Met residues in kinase substrate proteins, such as NR, can inhibit the phosphorylation of nearby sites and thereby couple oxidative signals to changes in protein phosphorylation

The Rho-Rock-Myosin Signaling Axis Determines Cell-Cell Integrity of Self-Renewing Pluripotent Stem Cells

Background: Embryonic stem (ES) cells self-renew as coherent colonies in which cells maintain tight cell-cell contact. Although intercellular communications are essential to establish the basis of cell-specific identity, molecular mechanisms underlying intrinsic cell-cell interactions in ES cells at the signaling level remain underexplored. Methodology/Principal Findings: Here we show that endogenous Rho signaling is required for the maintenance of cell-cell contacts in ES cells. siRNA-mediated loss of function experiments demonstrated that Rock, a major effector kinase downstream of Rho, played a key role in the formation of cell-cell junctional assemblies through regulation of myosin II by controlling a myosin light chain phosphatase. Chemical engineering of this signaling axis by a Rock-specific inhibitor revealed that cell-cell adhesion was reversibly controllable and dispensable for self-renewal of mouse ES cells as confirmed by chimera assay. Furthermore, a novel culture system combining a single synthetic matrix, defined medium, and the Rock inhibitor fully warranted human ES cell self-renewal independent of animal-derived matrices, tight cell contacts, or fibroblastic niche-forming cells as determined by teratoma formation assay. Conclusions/Significance: These findings demonstrate an essential role of the Rho-Rock-Myosin signaling axis for the regulation of basic cell-cell communications in both mouse and human ES cells, and would contribute to advance i

Regulation of plant development in Arabidopsis

The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file.Title from title screen of research.pdf file (viewed on June 19, 2009)Thesis (Ph. D.) University of Missouri-Columbia 2008.[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT REQUEST OF AUTHOR.] The development of a complex organism, such as a plant, requires the function of multiple pathways that regulate various aspects of the developmental process. Here pathways that regulate various aspects of plant development are investigated. A regulatory module consisting of a NAC-domain transcription factor, CUC2, in which targeting by the miRNA family, miRNA164, is disrupted, was investigated. This disruption resulted in an overaccumulation of the CUC2 transcript. This disruption uncovers roles for this regulatory module in controlling lateral organ enlargement and patterning. This regulatory module is proposed to act as a global regulator of lateral organ patterning and enlargement as well as meristem maintenance. A novel function for a Mitogen-Activated Protein Kinase (MAPK) cascade in floral organ abscission is uncovered. Many plants use abscission as a means to shed unwanted organs or to release fruit and seeds at maturity. This process requires tight coordination through regulatory pathways, including the MKK4/MKK5 MAPK cascade. The DEVIL (DVL) family encodes predicted small proteins that all carry a conserved C-terminal motif. While it remains unclear if these small proteins play a role in signaling pathways, several other small protein families in plants have been shown to play such a role. Here investigations into the active gene product and possible genetic interactors are discussed.Includes bibliographical reference

Regulation of floral organ abscission in Arabidopsis thaliana

Abscission is a developmental program that results in the active shedding of infected or nonfunctional organs from a plant body. Here, we establish a signaling pathway that controls abscission in Arabidopsis thaliana from ligand, to receptors, to downstream effectors. Loss of function mutations in Inflorescence Deficient in Abscission (IDA), which encodes a predicted secreted small protein, the receptor-like protein kinases HAESA (HAE) and HAESA-like 2 (HSL2), the Mitogen-Activated Protein Kinase Kinase 4 (MKK4) and MKK5, and a dominant-negative form of Mitogen-Activated Protein Kinase 6 (MPK6) in a mpk3 mutant background all have abscission-defective phenotypes. Conversely, expression of constitutively active MKKs rescues the abscission-defective phenotype of hae hsl2 and ida plants. Additionally, in hae hsl2 and ida plants, MAP kinase activity is reduced in the receptacle, the part of the stem that holds the floral organs. Plants overexpressing IDA in a hae hsl2 background have abscission defects, indicating HAE and HSL2 are epistatic to IDA. Taken together, these results suggest that the sequential action of IDA, HAE and HSL2, and a MAP kinase cascade regulates the programmed separation of cells in the abscission zone

In search of automatic rhythm analysis methods for Turkish and Indian art music

The aim of this paper is to identify and discuss various methods in computational rhythm description of Carnatic and Hindustani music of India, and Makam music of Turkey. We define and describe three relevant rhythm annotation tasks for these cultures—beat tracking, meter estimation, and downbeat detection. We then evaluate several methodologies from the state of the art in Music Information Retrieval (MIR) for these tasks, using manually annotated datasets of Turkish and Indian music. This evaluation provides insights into the nature of rhythm in these cultures and the challenges to automatic rhythm analysis. Our results indicate that the performance of evaluated approaches is not adequate for the presented tasks, and that methods that are suitable to tackle the culture specific challenges in computational analysis of rhythm need to be developed. The results from the different analysis methods enable us to identify promising directions for an appropriate exploration of rhythm analysis in Turkish, Carnatic and Hindustani music.This work is partly supported by the European Research Council under the European Union’s Seventh Framework Program, as part of the CompMusic project (ERC grant agreement 267583)