Proteomic Analysis of Arabidopsis pldα1 Mutants Revealed an Important Role of Phospholipase D Alpha 1 in Chloroplast Biogenesis

Phospholipase D alpha 1 (PLDα1) is a phospholipid hydrolyzing enzyme playing multiple regulatory roles in stress responses of plants. Its signaling activity is mediated by phosphatidic acid (PA) production, capacity to bind, and modulate G-protein complexes or by interaction with other proteins. This work presents a quantitative proteomic analysis of two T-DNA insertion pldα1 mutants of Arabidopsis thaliana. Remarkably, PLDα1 knockouts caused differential regulation of many proteins forming protein complexes, while PLDα1 might be required for their stability. Almost one third of differentially abundant proteins (DAPs) in pldα1 mutants are implicated in metabolism and RNA binding. Latter functional class comprises proteins involved in translation, RNA editing, processing, stability, and decay. Many of these proteins, including those regulating chloroplast protein import and protein folding, share common functions in chloroplast biogenesis and leaf variegation. Consistently, pldα1 mutants showed altered level of TIC40 (a major regulator of protein import into chloroplast), differential accumulation of photosynthetic protein complexes and changed chloroplast sizes as revealed by immunoblotting, blue-native electrophoresis, and microscopic analyses, respectively. Our proteomic analysis also revealed that genetic depletion of PLDα1 also affected proteins involved in cell wall architecture, redox homeostasis, and abscisic acid signaling. Taking together, PLDα1 appears as a protein integrating cytosolic and plastidic protein translations, plastid protein degradation, and protein import into chloroplast in order to regulate chloroplast biogenesis in Arabidopsis

Apoplast proteome reveals that extracellular matrix contributes to multistress response in poplar

<p>Abstract</p> <p>Background</p> <p>Riverine ecosystems, highly sensitive to climate change and human activities, are characterized by rapid environmental change to fluctuating water levels and siltation, causing stress on their biological components. We have little understanding of mechanisms by which riverine plant species have developed adaptive strategies to cope with stress in dynamic environments while maintaining growth and development.</p> <p>Results</p> <p>We report that poplar (<it>Populus </it>spp.) has evolved a systems level "stress proteome" in the leaf-stem-root apoplast continuum to counter biotic and abiotic factors. To obtain apoplast proteins from <it>P. deltoides</it>, we developed pressure-chamber and water-displacement methods for leaves and stems, respectively. Analyses of 303 proteins and corresponding transcripts coupled with controlled experiments and bioinformatics demonstrate that poplar depends on constitutive and inducible factors to deal with water, pathogen, and oxidative stress. However, each apoplast possessed a unique set of proteins, indicating that response to stress is partly compartmentalized. Apoplast proteins that are involved in glycolysis, fermentation, and catabolism of sucrose and starch appear to enable poplar to grow normally under water stress. Pathogenesis-related proteins mediating water and pathogen stress in apoplast were particularly abundant and effective in suppressing growth of the most prevalent poplar pathogen <it>Melampsora</it>. Unexpectedly, we found diverse peroxidases that appear to be involved in stress-induced cell wall modification in apoplast, particularly during the growing season. Poplar developed a robust antioxidative system to buffer oxidation in stem apoplast.</p> <p>Conclusion</p> <p>These findings suggest that multistress response in the apoplast constitutes an important adaptive trait for poplar to inhabit dynamic environments and is also a potential mechanism in other riverine plant species.</p

High Lift and Flow Separation Control Via Moving Wall Effects: an Experimental and Numerical Investigation

A wing was designed with a moving surface high-lift device in the form of a rotating cylinder at the leading edge to improve low speed flight characteristics. This rotating cylinder accelerates the air flow over the top of the wing, effectively combining the concept of lift generated by an airfoil and lift generated by a rotating cylinder. This faster moving air over the top of the wing increases the pressure differential, thus increasing lift. The added momentum to the air flow results in delayed flow separation and a decrease in drag. For experimental testing, a wing was built using balsawood, basswood and MonoKote and was tested in a subsonic wind tunnel using two different tests stands. For validation and further testing, the high-lift device was modeled in Gambit and numerical simulations were performed using ANSYS Fluent. Experimental and numerical data show the high-lift device to be effective

Fundamental Predictions of Ion Intensities in Tandem Mass Spectra of Biomolecules

Tandem mass spectrometry (MSMS) has become a powerful tool for the analysis of biomolecules. To reveal molecular identity, experimental mass (m/z) data are either matched to appropriate databases or processed de-novo. Both approaches are essentially one-dimensional, because the m/z values of fragment ions play dominant role, while the intensities (the second dimension) are being neglected, despite their potential to corroborate or contradict the identification. Unlike the trivial case of m/z values, predicting the ion intensities has not been mastered yet beyond empirical observation and statistical treatment. This dissertation presents a fundamental, structure-based algorithm for the prediction of fragment ion intensities in MSMS spectra of peptides and metabolites. The algorithm builds on the central hypothesis that the fragment ion intensities reflect the relative abundances of respective protonated precursor isomers prior to fragmentation. The hypothesis is supported by extensive experimental evidence showing that in ion trap mass detectors, relative ion intensities do not depend on the energy or activation time of fragmentation when commonly used ranges of conditions are explored. The multi-step algorithm developed includes molecular mechanics Monte Carlo conformational space sampling, semi-empirical calculations, and Density Functional Theory (DFT) quantum chemistry computations for structure refinement and energy calculations. A Boltzmann distribution determined from energy values of pertinent precursors accurately corresponded with relative ion intensities in MSMS spectra of three model pentapeptides. Reproducibility of the algorithm was tested, and while substantial differences were revealed among the multiple Monte Carlo samplings started from the same initial structures, the inconsistency was mitigated in following semi-empirical and DFT steps. The algorithm was optimized for efficiency as well. Computational costs were lowered (by more than 50%) by narrowing the energy window in which the conformers were taken to the following steps in the algorithm and finally to the Boltzmann distribution. For metabolites, ion intensity orders for nine out of eleven molecules were predicted correctly. However, the accuracy of the prediction of relative ion intensities was not satisfactory. Nevertheless, predicting the most intensive ion alone could be invaluable for preliminary metabolite identification and selecting good candidate standards for ultimate identification based on matched properties of analyte and standard

Maize-Pathogen Interactions: An Ongoing Combat from a Proteomics Perspective

Maize (Zea mays L.) is a host to numerous pathogenic species that impose serious diseases to its ear and foliage, negatively affecting the yield and the quality of the maize crop. A considerable amount of research has been carried out to elucidate mechanisms of maize-pathogen interactions with a major goal to identify defense-associated proteins. In this review, we summarize interactions of maize with its agriculturally important pathogens that were assessed at the proteome level. Employing differential analyses, such as the comparison of pathogen-resistant and susceptible maize varieties, as well as changes in maize proteomes after pathogen challenge, numerous proteins were identified as possible candidates in maize resistance. We describe findings of various research groups that used mainly mass spectrometry-based, high through-put proteomic tools to investigate maize interactions with fungal pathogens Aspergillus flavus, Fusarium spp., and Curvularia lunata, and viral agents Rice Black-streaked Dwarf Virus and Sugarcane Mosaic Virus

Shot-Gun Proteomic Analysis on Roots of Arabidopsis pldα1 Mutants Suggesting the Involvement of PLDα1 in Mitochondrial Protein Import, Vesicular Trafficking and Glucosinolate Biosynthesis

Phospholipase D&alpha;1 (PLD&alpha;1) belongs to phospholipases, a large phospholipid hydrolyzing protein family. PLD&alpha;1 has a substrate preference for phosphatidylcholine leading to enzymatic production of phosphatidic acid, a lipid second messenger with multiple cellular functions. PLD&alpha;1 itself is implicated in biotic and abiotic stress responses. Here, we present a shot-gun differential proteomic analysis on roots of two Arabidopsis pld&alpha;1 mutants compared to the wild type. Interestingly, PLD&alpha;1 deficiency leads to altered abundances of proteins involved in diverse processes related to membrane transport including endocytosis and endoplasmic reticulum-Golgi transport. PLD&alpha;1 may be involved in the stability of attachment sites of endoplasmic reticulum to the plasma membrane as suggested by increased abundance of synaptotagmin 1, which was validated by immunoblotting and whole-mount immunolabelling analyses. Moreover, we noticed a robust abundance alterations of proteins involved in mitochondrial import and electron transport chain. Notably, the abundances of numerous proteins implicated in glucosinolate biosynthesis were also affected in pld&alpha;1 mutants. Our results suggest a broader biological involvement of PLD&alpha;1 than anticipated thus far, especially in the processes such as endomembrane transport, mitochondrial protein import and protein quality control, as well as glucosinolate biosynthesis

A remodeled RNA polymerase II complex catalyzing viroid RNA-templated transcription.

Viroids, a fascinating group of plant pathogens, are subviral agents composed of single-stranded circular noncoding RNAs. It is well-known that nuclear-replicating viroids exploit host DNA-dependent RNA polymerase II (Pol II) activity for transcription from circular RNA genome to minus-strand intermediates, a classic example illustrating the intrinsic RNA-dependent RNA polymerase activity of Pol II. The mechanism for Pol II to accept single-stranded RNAs as templates remains poorly understood. Here, we reconstituted a robust in vitro transcription system and demonstrated that Pol II also accepts minus-strand viroid RNA template to generate plus-strand RNAs. Further, we purified the Pol II complex on RNA templates for nano-liquid chromatography-tandem mass spectrometry analysis and identified a remodeled Pol II missing Rpb4, Rpb5, Rpb6, Rpb7, and Rpb9, contrasting to the canonical 12-subunit Pol II or the 10-subunit Pol II core on DNA templates. Interestingly, the absence of Rpb9, which is responsible for Pol II fidelity, explains the higher mutation rate of viroids in comparison to cellular transcripts. This remodeled Pol II is active for transcription with the aid of TFIIIA-7ZF and appears not to require other canonical general transcription factors (such as TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, and TFIIS), suggesting a distinct mechanism/machinery for viroid RNA-templated transcription. Transcription elongation factors, such as FACT complex, PAF1 complex, and SPT6, were also absent in the reconstituted transcription complex. Further analyses of the critical zinc finger domains in TFIIIA-7ZF revealed the first three zinc finger domains pivotal for RNA template binding. Collectively, our data illustrated a distinct organization of Pol II complex on viroid RNA templates, providing new insights into viroid replication, the evolution of transcription machinery, as well as the mechanism of RNA-templated transcription

Vesicular Trafficking and Stress Response Coupled to PI3K Inhibition by LY294002 as Revealed by Proteomic and Cell Biological Analysis

LY294002 is a synthetic quercetin-like compound, which, unlike wortmannin, is more specific inhibitor of phosphatidylinositol 3-kinase (PI3K). It inhibits endocytosis and vacuolar transport. We report here on the proteome-wide effects of LY294002 on <i>Arabidopsis</i> roots focusing on proteins involved in vesicular trafficking and stress response. At the subcellular level, LY294002 caused swelling and clustering of late endosomes leading to inhibition of vacuolar transport. At the proteome level, this compound caused changes in abundances of proteins categorized to 10 functional classes. Among proteins involved in vesicular trafficking, a small GTPase ARFA1f was more abundant, indicating its possible contribution to the aggregation and fusion of late endosomes triggered by LY294002. Our study provides new information on storage proteins and vacuolar hydrolases in vegetative tissues treated by LY294002. Vacuolar hydrolases were downregulated, while storage proteins were more abundant, suggesting that storage proteins were protected from degradation in swollen multivesicular bodies upon LY294002 treatment. Upregulation of 2S albumin was validated by immunoblotting and immunolabeling analyses. Our study also pointed to the control of antioxidant enzyme machinery by PI3K because LY294002 downregulated two isozymes of superoxide dismutase. This most likely occurred via PI3K-mediated downregulation of protein AtDJ1A. Finally, we discuss specificity differences of LY294002 and wortmannin against PI3K, which are reflected at the proteome level. Compared with wortmannin, LY294002 showed more narrow and perhaps also more specific effects on proteins, as suggested by gene ontology functional annotation