1,720,985 research outputs found
Dissecting stimulus-specific Ca2+ signals in amyloplasts and chloroplasts of Arabidopsis thaliana cell suspension cultures
Full text linkCalcium is used by plants as an intracellular messenger in the detection of and response to a plethora of environmental stimuli and contributes to a fine-tuned internal regulation. Interest in the role of different subcellular compartments in Ca(2+) homeostasis and signalling has been growing in recent years. This work has evaluated the potential participation of non-green plastids and chloroplasts in the plant Ca(2+) signalling network using heterotrophic and autotrophic cell suspension cultures from Arabidopsis thaliana plant lines stably expressing the bioluminescent Ca(2+) reporter aequorin targeted to the plastid stroma. Our results indicate that both amyloplasts and chloroplasts are involved in transient Ca(2+) increases in the plastid stroma induced by several environmental stimuli, suggesting that these two functional types of plastids are endowed with similar mechanisms for handling Ca(2+). A comparison of the Ca(2+) trace kinetics recorded in parallel in the plastid stroma, the surface of the outer membrane of the plastid envelope, and the cytosol indicated that plastids play an essential role in switching off different cytosolic Ca(2+) signals. Interestingly, a transient stromal Ca(2+) signal in response to the light-to-dark transition was observed in chloroplasts, but not amyloplasts. Moreover, significant differences in the amplitude of specific plastidial Ca(2+) changes emerged when the photosynthetic metabolism of chloroplasts was reactivated by light. In summary, our work highlights differences between non-green plastids and chloroplasts in terms of Ca(2+) dynamics in response to environmental stimuli
The involvement of chloroplast Ca2+-permeable channels in plant organellar Ca2+ signalling
No full textThe role of calcium signaling in plants: from Arabidopsis to barley
Full text linkCa2+ is an important intracellular secondary messenger involved in many signal transduction pathways in plants. During abiotic and biotic stresses, specific Ca2+ signatures are formed within the plant cell causing a spike in the Ca2+ concentration that have a particular frequency, shape, and duration. Studying these Ca2+ signatures in detail will give a closer look into the plant's defense mechanisms caused by different gene regulations. Although, a lot of research on cytosolic and organellar Ca2+ transients has been performed in Arabidopsis thaliana in response to various stimuli, very little is known about the Ca2+ transients in crops such as Hordeum vulgare (barley). Therefore, the main aim of this project was to study the cytosolic Ca2+ transients caused in barley in response to oxidative and drought stress. For the first time, in this study, it was possible to target apoaequorin into the cytosol of barley to monitor the Ca2+ transients. The results showed stimulus specific Ca2+ signatures in barley and these signatures were compared with Arabidopsis. It was also revealed that these Ca2+ transients are tissue specific and dependent on the age of the barley seedling as well. By using inhibitors of the Ca2+ transients, it could be seen that the Ca2+ transients were caused by the influx of Ca2+ across the plasma membrane and from the Ca2+ release from internal stores like the endoplasmic reticulum. Furthermore, to investigate the gene expression involved in causing the Ca2+ transients in response to oxidative stress, RNA-sequencing was done using the leaf and root samples in the presence and absence of the Ca2+ channel inhibitor, lanthanum chloride. The results showed that there is a clear differentiation between stress responses that require a Ca2+ signal and those that are Ca2+ independent.
Ca2+ sensors, such as calmodulin (CaM) and CaM-like proteins (CML) transduce Ca2+ signals into a cellular response which are activated by different target proteins. In the second part of this work, a previously identified CaM/Ca2+ binding target protein called the Rieske iron-sulfur protein (RISP) was analyzed for its functionality and topology. To that end, the Ca2+-dependent CaM binding property of RISP was confirmed and the localization of the N- and C- terminus of the protein was found to be in the mitochondrial matrix indicating that the CaM/Ca2+ regulation occurs within the matrix. However, there still exists a possibility that the localization of the N-terminus of RISP is not exclusively fixed to the matrix but also to the inner membrane space
Molecular insights into abiotic stress responses in barley and Arabidopsis
Full text linkThis cumulative thesis, titled "Molecular insights into abiotic stress responses in barley and Arabidopsis", investigates two critical aspects of plant responses to abiotic stress. The first part explores hydrogen peroxide (H2O2) and its interaction with calcium (Ca2+) signalling in barley (Hordeum vulgare L.), while the second part examines drought stress regulation in Arabidopsis thaliana, revealing a novel mechanism involving the genes GASA3 and AFP1.
H2O2 plays a pivotal role in signalling pathways that enable plants to adapt to environmental challenges. Despite its importance, its transcriptomic impact remains underexplored. To address this, RNA-Seq analysis was used to examine changes in gene expression in barley roots and leaves after H2O2 treatment. This revealed 1883 differentially expressed genes (DEGs) in roots and 1001 in leaves, with most responses being tissue-specific. Only 209 DEGs were commonly regulated, and 37 showed opposing regulation across tissues. Gene ontology (GO) analysis highlighted the organ-specific nature of the response: leaf DEGs were enriched in hormone signalling, H2O2 response, and abiotic stress adaptation, while root DEGs were associated with H2O2 detoxification, glutathione metabolism, and cell wall modifications. A follow-up study examined the cross-talk between H2O2 and Ca2+ signalling using RNA-Seq under conditions that blocked Ca2+-transients. By comparing expression profiles from H2O2-only and LaCl3+H2O2 treatments, 331 Ca2+-dependent H2O2-responsive genes in leaves and 1320 in roots were identified and grouped into five and four clusters, respectively. A SKM network analysis further revealed transcription factors potentially governing this H2O2–Ca2+ cross-talk.
Drought is one of the most severe abiotic stresses impacting plant growth, development, and reproduction. Like H2O2, drought triggers extensive transcriptomic reprogramming. In this study, two strongly drought-induced genes, GASA3 and AFP1, were identified. Loss-of-function mutants showed enhanced drought resistance, while constitutive overexpression of either gene led to reduced tolerance. The gasa3afpl double mutant exhibited even greater resistance than single mutants. Both genes are also ABA-inducible, though GASA3 expression remained low in the absence of AFP1. The improved drought tolerance in mutants was linked to higher leaf water content due to smaller stomatal apertures and reduced transpiration. Additionally, ABA levels were elevated in mutants under drought stress- not due to increased biosynthesis, but via the release of conjugated vacuolar ABA-GE through β-glucosidase BG2. Consistent with this, ABA-responsive genes such as RD29A/B and ABF2/3 were more strongly upregulated in mutants than in wild type (WT). Conversely, PP2CA, which encodes a phosphatase involved in ABA negative feedback, was repressed in the absence of GASA3 and AFP1. These results suggest that both genes act as negative regulators of drought tolerance, with AFP1 influencing GASA3 expression.
Altogether, this thesis offers new insights into plant abiotic stress responses and provides a foundation for future functional studies in barley and other crops, potentially guiding breeding strategies for improved stress resilience under changing climates
JAR1-mediated JA-Ile accumulation: a mechanism towards drought stress resistance in <em>Arabidopsis thaliana</em>
Full text linkIn the present work, the regulatory capacity of biologically active jasmonate, jasmonyl-isoleucine (JA-Ile), under normal and progressive drought stress conditions throughout a plant’s life-cycle was investigated. To alter endogenous JA-Ile levels, two different plant lines were used: i) the T-DNA insertion line jar1-11, which contains significantly reduced amount of JA-Ile and ii) complementary to this, a T-DNA insertion line (JAR1-OE) expressing JAR1.1-YFP under the control of the 35S promoter, which results in JAR1 overexpression and enhanced endogenous JA-Ile levels. This line was newly developed within this work. Both lines displayed difference in growth and stress resistance compared to the wild type and each other. Under normal growth conditions jar1-11 plants displayed a larger rosette with narrower leaf blades, while JAR1-OE plants had stunted growth with lateral leaves. And while JAR1-OE was late in flowering, a reciprocal trend was observed in jar1-11. Furthermore, jar1-11 plants were more susceptible to drought stress, while JAR1-OE plants were highly resistant. In line with the difference in JAR1, hormone analysis revealed increased accumulation of JA-Ile in JAR1-OE under drought, while jar1-11 accumulated JA that could not be converted to JA-Ile. In addition, the homeostasis of some precursors and highly abundant catabolic products of JA and JA-Ile were differentially affected in these lines. Global gene expression analysis by RNA-seq revealed a reprogramming of the jasmonate signaling pathway with a positive feedback upregulation in JAR1-OE under drought stress. By contrast, in jar1-11 the biosynthesis of jasmonates was inhibited. Positive feedback in JAR1-OE helps plants to acquire pre-stress tolerance with positive stomatal regulation, anti-oxidant activity and modulation of ABA biosynthesis. This ultimately helps the plants in coping with subsequent drought stress through regulation of the photosynthetic machinery and other biological processes. Furthermore, calmodulin-like protein 12 (CML12) was identified as a potential target of jasmonate signaling. Intriguingly, CML12 behaves differentially at the transcriptional and translational levels to the presence or absence of JAR1 or endogenously added JA-Ile supporting a potential cross-talk between jasmonate and Ca2+-signaling. Finally, the transcription factor AtMYB2 was found to be a regulator of jasmonate signaling as it could control the accumulation of JA and JA-Ile under normal growth as well as drought stress conditions
Keeping in Touch: Visualising and Perturbing Photorespiratory Organelle Proximity in Plants
No full textOrganelles within cells are typically depicted as isolated structures, but increasing evidence shows that they are interconnected via specific membrane contact sites (MCS). While MCS research in mammals and yeast is gaining importance, MCS are less explored in plants. Photorespiration is ametabolic process that occurs across three organelles in plants: chloroplasts, peroxisomes and mitochondria. Whereas this process is well studied on the molecular level, only little is known about the relevance of inter-organellar contacts. The aim of this study was to elucidate the significance of organellar interactions and to determine how MCS are linked to plant growth and performance with the photorespiratory organelles as a model. We addressed this question with three main experimental approaches: 1) quantifying the proximity between chloroplasts and peroxisomes under different photosynthetic conditions, 2) testing potential dynamic or irreversible reporter systems for organelle proximity, and 3) manipulating the spatial organisation of cells by introducing a synthetic tether construct.
Previous reports evidenced physical associations and an increased interaction rate between the photorespiratory organelles in response to light in A. thaliana. We developed an automated high-throughput Python-based analysis pipeline for the quantification of organelle proximity. We used confocal z-stacks of cells with fluorescently labelled organelles and performed analyses in three model plant species. We were not able to replicate the findings of previous reports using manual image analysis or the Python-based analysis pipeline, potentially due to minor but critical changes in the experimental setup.
Secondly, we tested potential fluorescence-based proximity reporters, based on Bimolecular Fluorescence Complementation (BiFC) or Forster Resonance Energy Transfer/Fluorescence Lifetime Imaging (FRET/FLIM).We successfully targeted the potential proximity reporters to the cytosolic face of the photorespiratory organelles. Using splitYFP-based proximity sensors, we found unspecific homogeneous organellar membrane labelling, whereas the investigation of organelle positioning revealed tethering between peroxisomes and chloroplasts. Moreover, we created an inducible 2in1 gateway vector system(pInd) to test the suitability of self-assembling GFP (saGFP) and to enable inducible expression in transgenic lines. Testing the expression of a saGFP-based proximity sensor between chloroplasts and peroxisomes transiently in N. tabacum, we found a peroxisomal membrane labelling with increased GFP signal at putative MCS. Using FRET/FLIM-based proximity sensors, controls mimicking 100% and no organellar interaction were established, while dynamic imaging did not reveal a decrease in fluorescence lifetime at putative MCS between chloroplasts and peroxisomes.
The third approach involved introducing a synthetic tether to disturb the spatial organisation of the photorespiratory organelles. We were able to obtain transgenic A. thaliana lines showing curly leaves, impaired growth, an accelerated senescence, and a reduced high light tolerance including decreased anthocyanin accumulation. On cellular level, the overexpression of the synthetic tether resulted in the formation of spherical peroxisomal clusters, where mitochondrial structures also accumulated
The CAS protein and Ca<sup>2+</sup> signaling in the chloroplast of <em>Arabidopsis thaliana</em>
No full textThere has been evidence that CAS forms a complex in the chloroplast of Chlamydomonas with PSI, CYTb6/f, and other proteins like PGRL1 and ANR1. However, in A. thaliana, such a CAS complex has not been shown yet. Therefore, one of the purposes of this study was to investigate whether CAS can also build a protein complex in the chloroplast of A. thaliana. On the other hand, to better understand the role of CAS in A. thaliana, growth phenotype and photosynthetic activity were investigated in a cas-knockout (casko) A. thaliana mutant line compared to wild-type plants (WT).
CAS was also investigated as part of the Ca2+ signaling pathway in response to salt, mannitol, and oxidative stress. Using WT and casko mutant plants expressing the Ca2+ reporter AEQ, targeted to the cytosol, stroma, and thylakoid lumen, changes in [Ca2+] in response to these stimuli was examined. Additionally, this study has focused on identifying the potential sources of Ca2+ that may contribute to the change in [Ca2+].
Furthermore, we also sought to determine the correct cellular localization of CML36, one of the 50 CMLs found in the A. thaliana, by using the full-length YFP-tag and the self-assembly GFP (saGFP) system in leaf tissue and isolated protoplasts and using laser confocal fluorescence microscopy
Comparative analysis of wild-type and chloroplast MCU-deficient plants reveals multiple consequences of chloroplast calcium handling under drought stress
Full text linkIntroductionChloroplast calcium homeostasis plays an important role in modulating the response of plants to abiotic and biotic stresses. One of the greatest challenges is to understand how chloroplast calcium-permeable pathways and sensors are regulated in a concerted manner to translate specific information into a calcium signature and to elucidate the downstream effects of specific chloroplast calcium dynamics. One of the six homologs of the mitochondrial calcium uniporter (MCU) was found to be located in chloroplasts in the leaves and to crucially contribute to drought- and oxidative stress-triggered uptake of calcium into this organelle.MethodsIn the present study we integrated comparative proteomic analysis with biochemical, genetic, cellular, ionomic and hormone analysis in order to gain an insight into how chloroplast calcium channels are integrated into signaling circuits under watered condition and under drought stress.ResultsAltogether, our results indicate for the first time a link between chloroplast calcium channels and hormone levels, showing an enhanced ABA level in the cmcu mutant already in well-watered condition. Furthermore, we show that the lack of cMCU results in an upregulation of the calcium sensor CAS and of enzymes of chlorophyll synthesis, which are also involved in retrograde signaling upon drought stress, in two independent KO lines generated in Col-0 and Col-4 ecotypes.ConclusionsThese observations point to chloroplasts as important signaling hubs linked to their calcium dynamics. Our results obtained in the model plant Arabidopsis thaliana are discussed also in light of our limited knowledge regarding organellar calcium signaling in crops and raise the possibility of an involvement of such signaling in response to drought stress also in crops
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