Proteomanalyse von Pflanzen

Titelseite Inhaltsverzeichnis 1. Einleitung 1 2\. Methoden 13 3\. Ergebnisse 46 4\. Diskussion 99 5\. Zusammenfassung 121 6\. Summary 122 7\. Abkuerzungen 123 8\. Literaturverzeichnis 124 9\. Eigene Veroeffentlichungen 138 10\. Danksagung 140Within the course of this PhD thesis twodimensional gelelectrophoresis and MALDI-TOF-MS based methods have been evaluated and developed to fulfil the aim of large scale plant proteomics. Particular attention was paid to the optimisation of proteinextraction, proteinseparation and proteinstaining. In this context a new fractionation based proteinextraction method which gave rise to an 300 % increased display of proteinspots on 2-DE gels could be established. On the basis of the developed techniques we were able to establish a set of 2-DE standardpatterns from 8 different Arabidopsis thaliana tissues. Apart from the optimisation of 2-DE techniques an improved, robust and automated MALDI sample preparation system could be established. The set up of these methods allows the analysis and handling of more than 1000 proteinspots from 2-DE gels per day. In a following step the combination of the 2-DE-and the MALDI protocols will be employed for the large scale identification of a vast portion of the Arabidopsis thaliana proteome. As a primary step it was possible in a set of proof of principle experiments to identify 681 proteinspots from two different Arabidopsis thaliana leaf fraction 2-DE gels. Further we identified 352 proteinspots from an Arabidopsis thaliana silique 2-DE gel. In total the number of these preliminary identified proteins exceeds by far the number of previous published 2-DE proteomic data from Arabidopsis thaliana. In a second proof of principle experiment it was possible to show the increased separation capabilities of 2-DE gels. In this example the identification of differentially expressed proteins from water starved cucumber plants was achieved. This experiment clearly showed the need of two-dimensional separation of proteins from complex mixtures to display differentially expressed proteins, since one-dimensional protein separation is not sufficient to fulfil this task. In a last example the combination of tissue prefractionation techniques with 2-DE and MALDI-MS was used to identify an Arabidopsis thaliana subproteome. In this case the purification of cytosolic 80S ribosomal proteins was achieved by sucrose gradient density centrifugation of Arabidopsis thaliana leaf tissue. In this experiment it was possible to identify a large part (70 %) of the expected protein components of plants cytosolic ribosome from a single gel in a single round of identification. In total a number of 224 proteinspots could be identified from this ribosomal sample.Im Rahmen der vorliegenden Arbeit wurden Techniken ebenso wie Methoden evaluiert und entwickelt, die basierend auf der Verwendung von zweidimensionaler Gelelektrophorese und MALDI-TOF-MS Proteomanalysen von pflanzlichen Geweben ermöglichen. Hierbei wurden zunächst optimierte Methoden für die Proteinextraktion, Proteintrennung und die Proteinfärbung für die 2-DE erarbeitet. So konnte z. B. eine neue fraktionierte Gesamtproteinextraktionsmethode für Pflanzengewebe entwickelt werden, die eine bis zu 300 % verbesserte Ausbeute an Proteinspots auf 2-DE-Gelen im Vergleich zu Standardextraktionsmethoden zulässt. Diese optimierten Methoden wurden in einem weiterführenden Schritt für die Herstellung von 2-DE-Standardmustern aus 8 unterschiedlichen Arabidopsis thaliana-Geweben eingesetzt. Neben der Methodenentwicklung im 2-DE-Bereich wurden im Bereich der MALDI- Probenpräparation und der MALDI-Analyse stabile und automatisierte Protokolle für die Identifikation von 2-DE-Gel-getrennten Proteinen etabliert. Diese Protokolle ermöglichen es, mehr als 1000 Proteinspots pro Tag massenspektrometrisch zu analysieren. Um die reelle Anwendbarkeit und Tauglichkeit der entwickelten Methoden zu dokumentieren, wurden anhand dreier charakteristischer pflanzenspezifischer Fragestellungen Beispielexperimente durchgeführt. So ließen sich in einem ersten Ansatz 681 Proteinspots aus 2 unterschiedlichen Arabidopsis thaliana-Blattproteinextrakten und 352 Proteinspots aus einem Arabidopsis thaliana-Schotenproteinextrakt identifizieren. Die hierbei identifizierten Proteine, die aus nur 3 2-DE-Gelen stammten, übersteigen in ihrer Summe bereits alle bis dato publizierten Daten im Bereich der Arabidopsis thaliana-2-DE-basierten Proteomforschung. In einem zweiten Anwendungsexperiment wurden differentiell regulierte Phloemproteine aus trockengestressten Gurkenpflanzen in 2-DE-Gelen dargestellt und identifiziert. Hierbei zeigte sich, dass diese Art der differentiellen Analyse komplexerer Proteinmischungen das Auflösungsvermögen von herkömmlichen eindimensionalen Trennsystemen übersteigt und somit fast ausschließlich mittels zweidimensionalen Trennsystemen durchgeführt werden sollte. In einem letzten Anwendungsbeispiel, dessen Fokus auf der Analyse des cytosolischen 80S Ribosoms aus Arabidopsis thaliana-Blättern lag, konnte durch die Kombination einer Gewebsvorfraktionierung und der 2-DE ein Großteil (70 %) der bekannten Komponenten des cytosolischen Ribosoms bestimmt werden. Insgesamt ließen sich hierbei 224 Proteinspots aus einem einzigen 2-DE-Gel identifizieren

The Great Observatories Origins Deep Survey

The Great Observatories Origins Deep Survey (GOODS) is designed to gather the best and deepest multiwavelength data for studying the formation and evolution of galaxies and active galactic nuclei, the distribution of dark and luminous matter at high redshift, the cosmological parameters from distant supernovae, and the extragalactic background light. The program uses the most powerful space- and ground-based telescopes to cover two fields, each 10'x16', centered on the Hubble Deep Field North and the Chandra Deep Field South, already the sites of extensive observations from X-ray through radio wavelengths. GOODS incorporates 3.6-24 micron observations from a SIRTF Legacy Program, four-band ACS imaging from an HST Treasury Program, and extensive new ground-based imaging and spectroscopy. GOODS data products will be made available on a rapid time-scale, enabling community research on a wide variety of topics. Here we describe the project, emphasizing its application for studying the mass assembly history of galaxies.Comment: 8 pages, 2 figures, to appear in the proceedings of the ESO/USM Workshop "The Mass of Galaxies at Low and High Redshift" (Venice, Italy, October 2001), eds. R. Bender and A. Renzin

Galaxy Clustering at z~3

Galaxies at very high redshift (z~3 or greater) are now accessible to wholesale observation, making possible for the first time a robust statistical assessment of their spatial distribution at lookback times approaching ~90% of the age of the Universe. This paper summarizes recent progress in understanding the nature of these early galaxies, concentrating in particular on the clustering properties of photometrically selected ``Lyman break'' galaxies. Direct comparison of the data to predictions and physical insights provided by galaxy and structure formation models is particularly straightforward at these early epochs, and results in critical tests of the ``biased'', hierarchical galaxy formation paradigm.Comment: Presented at Royal Society Discussion Meeting, March 1998, "Large Scale Structure in the Universe", 14 pages LaTeX, 7 ps figures, uses rspublic.sty (included

The Stellar Masses and Star Formation Histories of Galaxies at z ≈ 6: Constraints from Spitzer Observations in the Great Observatories Origins Deep Survey

Using the deep Spitzer Infrared Array Camera (IRAC) observations of the Great Observatories Origins Deep Survey (GOODS), we study the stellar masses and star formation histories of galaxies at z approx 6 based on the i_(775)-band dropout sample selected from the GOODS fields. In total, we derive stellar masses for 53 i_(775)-band dropouts that have robust IRAC detections. These galaxies have typical stellar masses of ~10^(10) M_⊙ and typical ages of a couple of hundred million years, consistent with earlier results based on a smaller sample of z ≈ 6 galaxies. The existence of such massive galaxies at z ≈ 6 can be explained by at least one set of N-body simulations of the hierarchical paradigm. We also study 79 i_(775)-band dropouts that are invisible in the IRAC data and find that they are typically less massive by a factor of 10. These galaxies are much bluer than those detected by the IRAC, indicating that their luminosities are dominated by stellar populations with ages ≾ 40 Myr. Based on our mass estimates, we derive a lower limit to the global stellar mass density at z ≈ 6, which is 1.1-6.7 × 10^6 M_⊙ Mpc^(-3). The prospect of detecting the progenitors of the most massive galaxies at yet higher redshifts is explored. We also investigate the implication of our results for reionization and find that the progenitors of the galaxies comparable to those in our sample, even in the most optimized (probably unrealistic) scenario, cannot sustain the reionization for a period longer than ~2 Myr. Thus most of the photons required for reionization must have been provided by other sources, such as the progenitors of the dwarf galaxies that are far below our current detection capability

IRAC Deep Survey of COSMOS

Over the last four years, we have developed the COSMOS survey field with complete multi-wavelength coverage from radio to X-ray, including a total of 600 hours of Spitzer Legacy time (166 hours IRAC, 460 hours MIPS). Here we propose to deepen the IRAC 3.6 µm and 4.5 µm coverage with 3000 hours over 2.3 deg^2 area included in deep Subaru imaging. This extended mission deep survey will increase the sensitivity by a factor of 3–5. The most important impact will be that the COSMOS survey will then provide extremely sensitive photometric redshifts and stellar mass estimates for approximately a million galaxies out to z~6. We expect these data to detect approximately 1000 objects at z = 6 to 10. The data will also provide excellent temporal coverage for variability studies on timescales from days to the length of the extended mission

Probing Outflows in z= 1~2 Galaxies through FeII/FeII* Multiplets

We report on a study of the 2300-2600\AA FeII/FeII* multiplets in the rest-UV spectra of star-forming galaxies at 1.0<z<2.6 as probes of galactic-scale outflows. We extracted a mass-limited sample of 97 galaxies at z~1.0-2.6 from ultra-deep spectra obtained during the GMASS spetroscopic survey in the GOODS South field with the VLT and FORS2. We obtain robust measures of the rest equivalent width of the FeII absorption lines down to a limit of W_r>1.5 \AA and of the FeII* emission lines to W_r>0.5 \AA. Whenever we can measure the systemic redshift of the galaxies from the [OII] emission line, we find that both the FeII and MgII absorption lines are blueshifted, indicative that both species trace gaseous outflows. We also find, however, that the FeII gas has generally lower outflow velocity relative to that of MgII. We investigate the variation of FeII line profiles as a function of the radiative transfer properties of the lines, and find that transitions with higher oscillator strengths are more blueshifted in terms of both line centroids and line wings. We discuss the possibility that FeII lines are suppressed by stellar absorptions. The lower velocities of the FeII lines relative to the MgII doublet, as well as the absence of spatially extended FeII* emission in 2D stacked spectra, suggest that most clouds responsible for the FeII absorption lie close (3~4 kpc) to the disks of galaxies. We show that the FeII/FeII* multiplets offer unique probes of the kinematic structure of galactic outflows.Comment: 53 pages, 22 Figures, accepted for publication in ApJ, revised according to referee comment

Biases and Uncertainties in Physical Parameter Estimates of Lyman Break Galaxies from Broad-band Photometry

We investigate the biases and uncertainties in estimates of physical parameters of high-redshift Lyman break galaxies (LBGs), such as stellar mass, mean stellar population age, and star formation rate (SFR), obtained from broad-band photometry. By combining LCDM hierarchical structure formation theory, semi-analytic treatments of baryonic physics, and stellar population synthesis models, we construct model galaxy catalogs from which we select LBGs at redshifts z ~ 3.4, 4.0, and 5.0. The broad-band spectral energy distributions (SEDs) of these model LBGs are then analysed by fitting galaxy template SEDs derived from stellar population synthesis models with smoothly declining SFRs. We compare the statistical properties of LBGs' physical parameters -- such as stellar mass, SFR, and stellar population age -- as derived from the best-fit galaxy templates with the intrinsic values from the semi-analytic model. We find some trends in these distributions: first, when the redshift is known, SED-fitting methods reproduce the input distributions of LBGs' stellar masses relatively well, with a minor tendency to underestimate the masses overall, but with substantial scatter. Second, there are large systematic biases in the distributions of best-fit SFRs and mean ages, in the sense that single-component SED-fitting methods underestimate SFRs and overestimate ages. We attribute these trends to the different star formation histories predicted by the semi-analytic models and assumed in the galaxy templates used in SED-fitting procedure, and to the fact that light from the current generation of star-formation can hide older generations of stars. These biases, which arise from the SED-fitting procedure, can significantly affect inferences about galaxy evolution from broadband photometry.Comment: 85 pages, 34 figures, submittted to ApJ