Phytochrome control of plant growth and metabolism in Arabidopsis thaliana

Plants rely on light to supply photosynthetic energy and to provide information of the surrounding environment. Phytochromes are photoreceptors that sense external light quality and quantity, which in turn guide the strategy of plant growth. A large body of research has focused on Arabidopsis thaliana seedlings, where phytochrome control of responses such as hypocotyl elongation, hook opening and cotyledon greening, has been intensively explored. Mathematical models have also helped elucidate the molecular mechanism of phytochrome signalling. A smaller proportion of studies have investigated the role of phytochrome in controlling plant plasticity in adult plants. This work has shown that phytochrome depletion enhances leaf petiole elongation and slows growth, but there is a lack of information on how these marked changes alter metabolism. In this thesis, I use phytochrome multiple mutants of to explore how phytochromes interact with metabolism to affect plant growth. My analysis revealed that phytochrome loss results in dramatically reduced biomass production, especially in high order phyABDE mutant that lacks four out of five phytochromes. This is caused, at least partly, by impaired photosynthesis in phytochrome mutants, including reduced chlorophyll level and less CO2 uptake. Furthermore, cell wall synthesis and protein levels, major dry biomass constituents, are also repressed in phytochrome-depleted plants. Interestingly, these mutants accumulate more daytime sucrose and starch than wild type does, possibly due to their retarded growth in light. Further metabolic profiling reveals that these phytochrome mutants over-accumulate sugars, organic acids and amino acids. The sizable increase in raffinose and proline suggests a possible link to stress tolerance. Indeed, ABA and salt responses are significantly reduced in phytochrome mutants at both seedling and adult stages. These mutants are also more resistant to prolonged darkness, with less chlorophyll degradation in dark and higher survival rates. Collectively, this thesis shows that phytochromes have a novel role in plant resource management, controlling the allocation of resources for growth, switching the metabolism between growth and stress-coping states based on the availability of light from the environment. It brings new interest into phytochrome research in Arabidopsis, suggesting possible application of such knowledge to crop studies in the future

The circadian clock gene circuit controls protein and phosphoprotein rhythms in Arabidopsis thaliana

Twenty-four-hour, circadian rhythms control many eukaryotic mRNA levels, whereas the levels of their more stable proteins are not expected to reflect the RNA rhythms, emphasizing the need to test the circadian regulation of protein abundance and modification. Here we present circadian proteomic and phosphoproteomic time series from Arabidopsis thaliana plants under constant light conditions, estimating that just 0.4% of quantified proteins but a much larger proportion of quantified phospho-sites were rhythmic. Approximately half of the rhythmic phospho-sites were most phosphorylated at subjective dawn, a pattern we term the “phospho-dawn.” Members of the SnRK/CDPK family of protein kinases are candidate regulators. A CCA1-overexpressing line that disables the clock gene circuit lacked most circadian protein phosphorylation. However, the few phospho-sites that fluctuated despite CCA1-overexpression still tended to peak in abundance close to subjective dawn, suggesting that the canonical clock mechanism is necessary for most but perhaps not all protein phosphorylation rhythms. To test the potential functional relevance of our datasets, we conducted phosphomimetic experiments using the bifunctional enzyme fructose-6-phosphate-2-kinase/phosphatase (F2KP), as an example. The rhythmic phosphorylation of diverse protein targets is controlled by the clock gene circuit, implicating posttranslational mechanisms in the transmission of circadian timing information in plants

Phytochromes control metabolic flux, and their action at the seedling stage determines adult plant biomass

Phytochrome (phy) photoreceptors are known to regulate plastic growth responses to vegetation shade. However, recent reports also suggest an important role for phys in carbon resource management, metabolism, and growth. Here, we use 13CO2 labelling patterns in multi-allele phy mutants to investigate the role of phy in the control of metabolic fluxes. We also combine quantitative data of 13C incorporation into protein and cell wall polymers, gas exchange measurements and system modelling to investigate why biomass is decreased in adult multi-allele phy mutants. Phy influences the synthesis of stress metabolites like raffinose and proline, and the accumulation of sugars, possibly through regulating vacuolar sugar transport. Remarkably, despite their modified metabolism and vastly altered architecture, growth rates in adult phy mutants resemble those of wild-type plants. Our results point to delayed seedling growth and smaller cotyledon size as the cause of the adult-stage phy mutant biomass defect. Our data signify a role for phy in metabolic stress physiology, carbon partitioning and illustrate that phy action at the seedling stage sets the trajectory for adult biomass production

qpMerge: Merging different peptide isoforms using a motif centric strategy

Accurate quantification and enumeration of peptide motifs is hampered by redundancy in peptide identification. A single phosphorylation motif may be split across charge states, alternative modifications (e.g. acetylation and oxidation), and multiple miss-cleavage sites which render the biological interpretation of MS data a challenge. In addition motif redundancy can affect quantitative and statistical analysis and prevent a realistic comparison of peptide numbers between datasets. In this study, we present a merging tool set developed for the Galaxy workflow environment to achieve a non-redundant set of quantifications for phospho-motifs. We present a Galaxy workflow to merge three exemplar dataset, and observe reduced phospho-motif redundancy and decreased replicate variation. The qpMerge tools provide a straightforward and reusable approach to facilitating phospho-motif analysis. The source-code and wiki documentation is publically available at http://sourceforge.net/projects/ppmerge. The galaxy pipeline used in the exemplar analysis can be found at http://www.myexperiment.org/workflows/4186

Label-free quantitative analysis of the casein kinase 2-responsive phosphoproteome of the marine minimal model species Ostreococcus tauri

Casein kinase 2 (CK2) is a protein kinase that phosphorylates a plethora of cellular target proteins involved in processes including DNA repair, cell cycle control, and circadian timekeeping. CK2 is functionally conserved across eukaryotes, although the substrate proteins identified in a range of complex tissues are often different. The marine alga Ostreococcus tauri is a unicellular eukaryotic model organism ideally suited to efficiently study generic roles of CK2 in the cellular circadian clock. Overexpression of CK2 leads to a slow circadian rhythm, verifying functional conservation of CK2 in timekeeping. The proteome was analysed in wild‐type and CK2‐overexpressing algae at dawn and dusk, revealing that differential abundance of the global proteome across the day is largely unaffected by overexpression. However, CK2 activity contributed more strongly to timekeeping at dusk than at dawn. The phosphoproteome of a CK2 overexpression line and cells treated with CK2 inhibitor was therefore analysed and compared to control cells at dusk. We report an extensive catalogue of 447 unique CK2‐responsive differential phosphopeptide motifs to inform future studies into CK2 activity in the circadian clock of more complex tissues. All MS data have been deposited in the ProteomeXchange with identifier PXD000975 (http://proteomecentral.proteomexchange.org/dataset/PXD000975)

Multidimensional signals and analytic flexibility: Estimating degrees of freedom in human speech analyses

Recent empirical studies have highlighted the large degree of analytic flexibility in data analysis which can lead to substantially different conclusions based on the same data set. Thus, researchers have expressed their concerns that these researcher degrees of freedom might facilitate bias and can lead to claims that do not stand the test of time. Even greater flexibility is to be expected in fields in which the primary data lend themselves to a variety of possible operationalizations. The multidimensional, temporally extended nature of speech constitutes an ideal testing ground for assessing the variability in analytic approaches, which derives not only from aspects of statistical modeling, but also from decisions regarding the quantification of the measured behavior. In the present study, we gave the same speech production data set to 46 teams of researchers and asked them to answer the same research question, resulting insubstantial variability in reported effect sizes and their interpretation. Using Bayesian meta-analytic tools, we further find little to no evidence that the observed variability can be explained by analysts’ prior beliefs, expertise or the perceived quality of their analyses. In light of this idiosyncratic variability, we recommend that researchers more transparently share details of their analysis, strengthen the link between theoretical construct and quantitative system and calibrate their (un)certainty in their conclusions

Circadian abundance and modification of proteins in arabidopsis

Circadian clocks are endogenous pacemakers found in many organisms including plants, generating approximately 24h rhythms. Knowledge about the plant circadian clock plays a role for crop improvement. The plant circadian clock and its downstream outputs have been studied in detail by transcriptomics, however post-transcriptional and post-translational aspects are still to be researched. In addition, it has recently been shown that a protein modification remains rhythmic when rhythmic transcription is absent. This gives evidence for the existence of two oscillators: a transcription-translation feedback loop and a non-transcriptional oscillator. The aim of this PhD is to gain knowledge about circadian changes in abundance and phosphorylation of proteins as well as protein-protein interaction using the model plant Arabidopsis thaliana. I used high-throughput proteomics and phosphoproteomics methods to identify hundreds of phosposites that change in abundance in WT plants as well as dozens of proteins that exhibit circadian changes in their abundance. I also found significant temporal changes in protein phosphorylation in the transcriptionally arrhythmic mutant CCA1-Ox, albeit with dynamics different from the WT, demonstrating that without transcriptional rhythms, protein modification can still undergo rhythmic changes to some extent. In addition, I found reproducibly that the majority of changing phosphopeptides are most abundant at dawn and this is independent of the presence of a functional transcriptional oscillator. Roles of different kinases and affected phosphoproteins are discussed. I chose one of the rhythmically phosphorylated proteins, the bifunctional enzyme F2KP, for further functional experiments. In vitro experiments demonstrate that the rhythmic phosphosite is important for the activity of the enzyme. This is discussed in the light of circadian regulation of carbon metabolism. In addition to these studies on circadian protein abundance and modification, I investigated time-of-day dependent protein-protein interaction of the clock protein GIGANTEA (GI). Using an interaction proteomics timecourse, I identified about 100 potential new interactors of GI, some of which are candidates for links between diel timing and carbon metabolism. These results will help to generate hypotheses for explaining the surprising pleiotrophy of gi mutants