Constructing gene regulatory networks for long term photosynthetic light acclimation in Arabidopsis thaliana

Abstract Background Photosynthetic light acclimation is an important process that allows plants to optimize the efficiency of photosynthesis, which is the core technology for green energy. However, currently little is known about the molecular mechanisms behind the regulation of the photosynthetic light acclimation response. In this study, a systematic method is proposed to investigate this mechanism by constructing gene regulatory networks from microarray data of Arabidopsis thaliana. Methods The potential TF-gene regulatory pairs of photosynthetic light acclimation have been obtained by data mining of literature and databases. Following the identification of these potential TF-gene pairs, they have been refined using Pearson's correlation, allowing the construction of a rough gene regulatory network. This rough gene regulatory network is then pruned using time series microarray data of Arabidopsis thaliana via the maximum likelihood system identification method and Akaike's system order detection method to approach the real gene regulatory network of photosynthetic light acclimation. Results By comparing the gene regulatory networks under the PSI-to-PSII light shift and the PSII-to-PSI light shift, it is possible to identify important transcription factors for the different light shift conditions. Furthermore, the robustness of the gene network, in particular the hubs and weak linkage points, are also discussed under the different light conditions to gain further insight into the mechanisms of photosynthesis. Conclusions This study investigates the molecular mechanisms of photosynthetic light acclimation for Arabidopsis thaliana from the physiological level. This has been achieved through the construction of gene regulatory networks from the limited data sources and literature via an efficient computation method. If more experimental data for whole-genome ChIP-chip data and microarray data with multiple sampling points becomes available in the future, the proposed method will be improved on by constructing the whole-genome gene regulatory network. These advances will greatly improve our understanding of the mechanisms of the photosynthetic system.</p

The nucleotide-binding sub-proteome of mustard chloroplasts and its involvement in plastid redox signaling

The actual keen about redox signal development at the plastid photosynthetic apparatus, transmission and the reply to the signal was highlighted by the contribution of three review article to this work. Pfannschmidt et al. 2008 summarizes short and long term acclimation responses (STR and LTR respectively) to redox signals of the Plastoquinone (PQ) pool and the involvement of putative phosphorylation cascades and thioredoxins as well as the influence of the redox state on primary target genes in plastids and nucleus. Further on experimental approaches for the generation of a defined redox state at the photosynthetic electron transport (PET) chain was discussed. Dietzel et al. 2008 reviews the different types of retrograde signals between plastids and nucleus as well as the complexity and interaction of the signaling cascades and networks and in Pfalz et al. 2012 the environmental influences on gene expression and recent findings within plastid redox signaling were discussed. For a detailed investigation of the adaption of plastid gene expression responding to plastid redox signals the gene expression machinery of chloroplasts itself was studied. An experimental approach was used for the generation of a defined redox signal in mustard cotyledons, the following isolation of its chloroplasts and further on the nucleotide binding sub-proteome using heparin-Sepharose (HS) (Steiner et al. 2009; Schröter et al. 2010). The characterization and comparison of mustard cotyledons acclimated to redox signal inducing Light-qualities with Arabidopsis thaliana cotyledons was important for the integration of new findings within Sinapis alba into established models (Steiner et al. 2009). An effect on the transcriptional regulation of the two plastome-encoded genes psaAB and psbA was studied here concerning promoter recognition and specificity (Steiner et al. 2009). The impact of phosphorylation events on gene expression was surveyed and confirmed by determination of the phosphorylation state of the HS fractions, the endogenous kinase activity and the cooperative influence of kinase activity and thiol redox state on Chloroplast transcription (Steiner et al. 2009). HS proteins fractions contain a high degree of DNA and especially psaA and psbA binding proteins which were identified using mass spectrometry and Brassicales databases (Steiner et al. 2009; Schröter et al. 2010; Steiner et al. 2011). Special emphasis was on the analysis of the essential subunits of the plastid-encoded plastid RNA-polymerase (PEP) which was well to prepare by 2 dimensional (2D) blue native (BN) gel electrophoresis (Schröter et al. 2010; Steiner et al. 2011). The degree of proteins involved in gene expression was strongly increased by the use of a second chromatographic step with Phosphocellulose (PC) additional to HS (Schröter et al. 2014). Visualization and identification of this nucleotide binding sub-proteome was the aim of the last publication included into this work giving access to a precise view on the gene expression related proteome of mustard plastids (Schröter et al. 2014).Drei review Artikel beleuchten das aktuelle Wissen über die Redoxsignalentwicklung im plastidären Photosyntheseapparat, die Signalübermittlung und –beantwotung. Pfannschmidt et al. 2008 fasst Kurzzeitund Langzeitantworten auf Redoxsignale des Plastochinonpools zusammen und darüberhinaus die Einbeziehung von Phosphorylierungskaskaden und Thioredoxinen sowie den Einfluss des Redoxstatus auf primäre Zielgene in Plastiden und dem Zellkern. Desweiteren wurden experimentelle Ansätze für die Erzeugung eines definierten Redoxstatus in der photosynthetischen Elektronentransportkette diskutiert. Dietzel et al. 2008 fasst die verschiedenen Typen retrograder Signale zwischen Plastiden und Zellkern zusammen sowie die Komplexität und Interaktion der Signalkaskaden und –netzwerke und in Pfalz et al. 2012 werden die Umwelteinflüsse auf die Genexpression und aktuelle Erkenntnisse über Redoxsignale diskutiert. Für eine detailierte Untersuchung der Genexpressionsadaption als Antwort auf plastidäre Redoxsignale wurde die Genexpressionsmaschinerie der Chloroplasten direkt studiert. Ein definiertes Redoxsignal wurde in Senfkeimlingen generiert, anschließend die Chloroplasten und schließlich die nukleotidbindenden Proteine mittels Chromatographie über Heparinsepharose (HS) isoliert (Steiner et al. 2009; Schröter et al. 2010). Der Vergleich und die Charakterisierung der Senfkeimlinge, die an das Redoxsignal induzierende Licht akklimatisiert waren, mit Arabidopsis thaliana Keimlingen war wichtig für die Integration neuer Erkenntnisse über Sinapis alba in etablierte Modelle (Steiner et al. 2009). Der Effekt auf die transkriptionale Regulierung der zwei plastomkodierten Gene psaAB und psbA wurde hinsichtlich Promotererkennung und –spezifität untersucht (Steiner et al. 2009). Die Auswirkung von Phosphorylierungen auf die Genexpression wurde, durch die Bestimmung des Phosphorylierungsgrades der HS Fraktionen, der endogene Kinaseaktivität und des kooperativen Einflusses der Kinaseaktivität und des Thiolredoxstatus auf die Chloroplastentranskription, untersucht (Steiner et al. 2009). HS Fraktionen besitzen einen hohen Grad an DNA- und speziell psaA- und psbA-bindenden Proteinen, die durch Massenspektrometrie und Analyse mit Brassicales-Datenbanken identifiziert werden können (Steiner et al. 2009; Schröter et al. 2010; Steiner et al. 2011). Der Schwerpunkt lag bei der Analyse der essentiellen Untereinheiten der plastidenkodierten plastidären RNAPolymerase (PEP), die gut durch 2 dimensionale (2D) blue native (BN) Gelelektrophorese präpariert werden konnte (Schröter et al. 2010; Steiner et al. 2011). Der Anteil an Proteinen der Genexpression konnte durch eine zweite Chromatographie über Phosphocellulose (PC) zusätzlich zur HSChromatographie erzielt werden (Schröter et al. 2014). In der letzten Publikation dieser Arbeit geht es vorrangig um die Visualisierung und Identifizierung dieses nukleotidbindenden Teilproteoms, wodurch ein Zugang zu einem detaillierteren Einblick in das genexpressionsrelevante Proteom der Senfplastiden erzielt wurde (Schröter et al. 2014)

Phylogenomic analysis of the Chlamydomonas genome unmasks proteins potentially involved in photosynthetic function and regulation

Chlamydomonas reinhardtii, a unicellular green alga, has been exploited as a reference organism for identifying proteins and activities associated with the photosynthetic apparatus and the functioning of chloroplasts. Recently, the full genome sequence of Chlamydomonas was generated and a set of gene models, representing all genes on the genome, was developed. Using these gene models, and gene models developed for the genomes of other organisms, a phylogenomic, comparative analysis was performed to identify proteins encoded on the Chlamydomonas genome which were likely involved in chloroplast functions (or specifically associated with the green algal lineage); this set of proteins has been designated the GreenCut. Further analyses of those GreenCut proteins with uncharacterized functions and the generation of mutant strains aberrant for these proteins are beginning to unmask new layers of functionality/regulation that are integrated into the workings of the photosynthetic apparatus

Gene regulatory network inference : connecting plant biology and mathematical modeling

Plant responses to environmental and intrinsic signals are tightly controlled by multiple transcription factors (TFs). These TFs and their regulatory connections form gene regulatory networks (GRNs), which provide a blueprint of the transcriptional regulations underlying plant development and environmental responses. This review provides examples of experimental methodologies commonly used to identify regulatory interactions and generate GRNs. Additionally, this review describes network inference techniques that leverage gene expression data to predict regulatory interactions. These computational and experimental methodologies yield complex networks that can identify new regulatory interactions, driving novel hypotheses. Biological properties that contribute to the complexity of GRNs are also described in this review. These include network topology, network size, transient binding of TFs to DNA, and competition between multiple upstream regulators. Finally, this review highlights the potential of machine learning approaches to leverage gene expression data to predict phenotypic outputs

Training Memory: Exploring the Intersection of Plant Stress Signalling and DNA Methylation

Plants are sessile organisms living in a dynamic environment to which they must continually acclimatize in order to maximise their reproductive potential. This plasticity is achieved through many complex and intricate signalling pathways that allow for the continuous perception, response, and adjustments to new environmental stimuli. A growing body of evidence suggests that such pathways are not merely static but dynamic and can be primed following repeated activation, thus affecting enhanced responses to recurring stresses. Such examples of priming have led to a notion that plants have some capacity to form stress memories of past environmental perturbations. However, the full extent and nature of such memory, and the machinery involved to store and transmit these, remain enigmatic. One prospective mechanism is the involvement of heritable, yet rapid and reversible, chromatin marks that, theoretically, could be shaped by the environment to convey a regulatory effect on the expression of the underlying genotype, thus acting as an epigenetic layer of regulation. This thesis explores the potential intersection of stress signalling pathways and chromatin variation, specifically DNA methylation, to co-ordinate plant stress responses. First, mechanistic insights into the operation of a SAL1-PAP-XRN retrograde signalling pathway to fine-tune plant physiology under drought are presented. A key finding was that this pathway complements canonical ABA signalling to induce stomatal closure, thus minimising water-loss under water limited conditions. Furthermore, the SAL1-PAP-XRN pathway was found to effect chromatin patterns, specifically DNA methylation at short transposable elements. These observations implicate cross-talk with the RNA directed DNA methylation pathway, however, the exact mechanism for this interaction remains to be identified. Multiple investigations were performed to test for stress-induced changes in DNA methylation that could potentially regulate responses to recurring stress, thus conveying a memory. A transgenerational recurring drought stress experiment tested whether descendants of drought-exposed lineages displayed greater drought tolerance (transgenerational memory). For the majority of traits tested, including plant growth rate and drought survival, offspring from plant lineages exposed to successive generations of repeated drought stress performed comparably to those from control lineages. However, memory was demonstrated in the form of enhanced seed dormancy, in drought stressed lineages, that persisted at least one generation removed from stress. Whether this capacity for memory could be related to the type or severity of stress applied, or species examined, remains to be investigated further. The transgenerational drought experiment was paired with a recurring excess-light stress experiment to investigate memory within a generation. Not only did this treatment lead to priming of plant photosynthetic behaviour, indicative of a greater capacity to withstand abrupt increases in light intensity, but new leaves from stressed plants, developed in the absence of stress, also showed altered photosynthetic characteristics compared to unstressed counterparts. Such observations are consistent with the mitotic transmission of stress-induced traits. Given multiple demonstrations of memory, comparisons were made to unstressed controls to test for any correlating changes in DNA methylation that might explain the phenomena observed. However, in both experiments, observations of memory were found to be independent of large-scale conserved changes in DNA methylation discounting it as a conveyor of plant stress memories, under these conditions, raising questions regarding the mechanism(s) responsible for the examples of memory observed herein. Ultimately, this thesis systematically evaluates the notion that plants are able to form genuine memories, potentially underpinned by reversible chromatin marks, that may facilitate acclimation to local environments on a relatively rapid scale compared to the fixation of adaptive genetic polymorphisms. Any capacity for plant stress memories may provide avenues for further epigenomic based agronomic tools to improve crop stress tolerance. However, the nature of such memories observed here appear subtle and nuanced, and are forgotten beyond a generation. Further characterisation and mechanistic understanding of mitotic memory mechanisms, however, may still hold potential. It was also observed that stress signalling pathways can interact with those involved in chromatin modification, giving novel insight into their mechanistic functioning and the how the onset of stress may induce chromatin changes. Despite this potential, the DNA methylome was found to be relatively impervious to stress-induced changes and, thus, is an unlikely memory mechanism

The Transcriptional Landscape and Hub Genes Associated with Physiological Responses to Drought Stress in Pinus tabuliformis

Drought stress has an extensive impact on regulating various physiological, metabolic, and molecular responses. In the present study, the Pinus tabuliformis transcriptome was studied to evaluate the drought-responsive genes using RNA- Sequencing approache. The results depicted that photosynthetic rate and H2O conductance started to decline under drought but recovered 24 h after re-watering; however, the intercellular CO2 concentration (Ci) increased with the onset of drought. We identified 84 drought-responsive transcription factors, 62 protein kinases, 17 transcriptional regulators, and 10 network hub genes. Additionally, we observed the expression patterns of several important gene families, including 2192 genes positively expressed in all 48 samples, and 40 genes were commonly co-expressed in all drought and recovery stages compared with the control samples. The drought-responsive transcriptome was conserved mainly between P. tabuliformis and A. thaliana, as 70% (6163) genes had a homologous in arabidopsis, out of which 52% homologous (3178 genes corresponding to 2086 genes in Arabidopsis) were also drought response genes in arabidopsis. The collaborative network exhibited 10 core hub genes integrating with ABA-dependent and independent pathways closely conserved with the ABA signaling pathway in the transcription factors module. PtNCED3 from the ABA family genes had shown significantly different expression patterns under control, mild, prolonged drought, and recovery stages. We found the expression pattern was considerably increased with the prolonged drought condition. PtNCED3 highly expressed in all drought-tested samples; more interestingly, expression pattern was higher under mild and prolonged drought. PtNCED3 is reported as one of the important regulating enzymes in ABA synthesis. The continuous accumulation of ABA in leaves increased resistance against drought was due to accumulation of PtNCED3 under drought stress in the pine needles